Binding molecules with modified J-chain

- IGM Biosciences, Inc.

The present invention provides binding molecules that include an IgM, IgA, IgG/IgM or IgG/IgA antibody with a modified J-chain that includes a binding moiety that antagonizes a T-cell inhibitory signaling pathway, and their uses.

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Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a US National Stage Entry of PCT Application No. PCT/US2016/055053, filed Sep. 30, 2016, which claims priority benefit of the filing date of U.S. Provisional Patent Application Ser. No. 62/235,486, filed on Sep. 30, 2015, which are each hereby incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII file, named Sequence-Listing.TXT, is 399 Kb in size and was created on 4 Jun. 2022.

FIELD OF THE INVENTION

The present invention concerns binding molecules that comprise an IgM, IgA, IgG/IgM or IgG/IgA antibody with a J-chain modified to include a binding moiety that affects a T-cell signaling pathway, and their uses.

BACKGROUND OF THE INVENTION

J-chain is an acidic 15-kDa polypeptide, which is associated with pentameric IgM and dimeric IgA via disulfide bonds involving the penultimate cysteine residue in the 18-amino acid secretory tail-piece (tp) at the C-terminus of the IgM μ or IgA α heavy chain. The three disulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, and Cys 108 and 133, respectively. See, e.g., Frutiger et al. 1992, Biochemistry 31, 12643-12647. Structural requirements for incorporation of the J-chain into human IgM and IgA and for polymeric immunoglobulin assembly and association with the J-chain are reported by Sorensen et al. 2000, Int. Immunol. 12(1): 19-27 and Yoo et al. 1999, J. Biol. Chem. 274(47):33771-33777, respectively. Recombinant production of soluble J-chain in E coli is reported by Redwan et al. 2006, Human Antibodies 15:95-102.

Methods for making hybrid IgA/IgG and IgM/IgG antibodies are known in the art. Thus, recombinant production of hybrid IgA2/IgG1 antibodies is reported in Chintalacharuvu et al. 2001, Clin Immunol 101(1):21-31. It has been reported that addition of αtp or μtp at the end of IgG γ heavy chain facilitates polymerization and enhances effector function such as complement activation (Smith et al., J Immunol 1995, 154:2226-2236). The IgA/IgG hybrid antibodies possess properties of both IgA and IgG. Methods for recombinant production of IgM antibodies are also known in the art. E.g., Tchoudakova A, et al., High level expression of functional human IgMs in human PER.C6 cells. mAbs. 2009; 1(2):163-171.

Despite the advances made in the design of antibodies, there remains a need for modified antibodies with improved properties, such as improved affinity, specificity and/or avidity, as well as the ability to bind to multiple binding targets.

As the field has progressed, antibody function has been enhanced through creative means of protein engineering, such as to provide higher affinity, longer half-life, and/or better tissue distribution, as well as combination of small and large molecule technologies for increased focus of cell destruction via toxic payload delivery (e.g., antibody-drug conjugates). Another approach to improving antibody function takes advantage of the bivalent binding of the immunoglobulin G (IgG) structure which allows one IgG molecule to bind two antigens. Indeed, in certain applications, there exists good potential for asymmetric antibodies to exert useful functions by simultaneously binding two different target antigens. To address this need, a variety of constructs have been produced to yield a single molecule that can bind two different antigens, allowing for functions never before seen in nature. An example of this bi-specific approach is “blinatumomab” (MT103 or AMG103) which binds the CD3 and CD19 receptors, on T- and B-cells, respectively. This tethering of a cytotoxic T-cell to a cancerous B-cell, allows for effective treatment of B-cell leukemia.

The blockade of immune checkpoints has emerged as a promising area for the advancement of cancer treatment. Immune checkpoints refer to inhibitory signaling pathways that are encoded into the immune system, and which play a vital role in maintaining self-tolerance, as well as modulating the duration and amplitude of immune responses. See, e.g., Pardoll, Drew M. “The blockade of immune checkpoints in cancer immunotherapy.” Nature Reviews Cancer 12.4 (2012): 252-264; Postow, Michael A. et al., “Immune Checkpoint Blockade in Cancer Therapy,” J Clin Oncol. 2015 Jun. 10; 33(17):1974-82. doi: 10.1200/JCO.2014.59.4358.

Despite positive proof of concept results in preclinical models, investigators have reported that monoclonal IgG blocking antibodies directed against T-cell inhibitory signaling pathway components (for example, ipilimumab (Bristol-Myers Squibb) and tremelimumab (MedImmune/AstraZenica), both directed against CTLA4) have only achieved minimal efficacy results in a clinical setting. E.g., Postow et al., pp. 1-2. In addition, treatments involving monoclonal IgG antibodies have resulted in immune-related adverse events, such as dermatologic, GI, hepatic, endocrine and other inflammatory events. E.g., Id. at p. 4. As such, the use of monoclonal IgG antibodies in immune checkpoint blockade may be limited by the therapeutic index of such molecules, in that the dose of a monoclonal IgG antibody required to elicit the desired therapeutic effect also causes immune-related adverse events.

Accordingly, there is a need for binding molecules with increased avidity that will provide increased potency so that lower dosage levels can be used, thereby preventing the occurrence of immune-related adverse events, while still achieving effective blockade of T-cell inhibitory signaling pathways.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the recognition that the J-chain of an IgM or IgA antibody can be modified by introducing one or more binding moieties into a native J-chain sequence, and the modified J-chain can be introduced into IgM, IgA, IgG/IgM or IgG/IgA antibodies without compromising the functionality of the recipient antibody or the binding of the modified J-chain to its target. This allows the modified J-chain with binding moiety to interact with one set of target antigens, while the IgM, IgA, IgG/IgM or IgG/IgA antibody can interact with a different set of target antigens.

The invention is further based on the recognition that due to their multivalent nature, IgM, IgA, IgG/IgM or IgG/IgA antibodies can provide increased avidity between the antibody and a target antigen, thereby facilitating binding of antigens with low level expression and/or low binding affinity. Furthermore, the optional multi-specific nature of the IgM, IgA, IgG/IgM or IgG/IgA portion of the subject binding molecules allows binding between specific numbers and/or specific types of binding targets, thereby facilitating binding between specific combinations of antigen targets. The modified J-chain portion of the subject binding molecules provides additional opportunities for target binding, allowing the subject binding molecules to bind one or more targets via the IgM, IgA, IgG/IgM or IgG/IgA portion of the molecule, while simultaneously binding to one or more targets via a binding moiety on the J-chain.

Aspects of the invention include binding molecules comprising an IgM, IgA, IgG/IgM or IgG/IgA antibody with a modified J-chain, or an antigen binding fragment thereof, wherein the modified J-chain comprises a binding moiety that affects a T-cell signaling pathway. In some embodiments, a binding molecule according to claim 1, wherein the binding moiety antagonizes a T-cell inhibitory signaling pathway. In some embodiments, a binding moiety on the modified J-chain binds to a cell surface protein selected from the group consisting of: CTLA4, PD-1, TIM3, LAG3, BTLA, VISTA and TIGIT. In some embodiments, the IgM, IgA, IgG/IgM or IgG/IgA antibody antagonizes a T-cell inhibitory signaling pathway. In some embodiments, the antibody binds to a target selected from the group consisting of: PD-1, PD-L1, TIM3 and LAG3.

In some embodiments, an IgM, IgA, IgG/IgM or IgG/IgA antibody agonizes a T-cell stimulatory signaling pathway. In some embodiments, the antibody binds to a target selected from the group consisting of: CD137, OX40, CD40, GITR, CD27 and HVEM. In some embodiments, the IgM, IgA, IgG/IgM or IgG/IgA antibody binds to a low level expression target. In some embodiments, the low level expression target is selected from the group consisting of: EGFR, HER2, HER3, EpCAM, CEACAM, Gp100, MAGE1 and PD-L1. In some embodiments, the low level expression target is a cell surface protein on an epithelial cancer cell.

In some embodiments, an IgM, IgA, IgG/IgM or IgG/IgA antibody binds to a low affinity target. In some embodiments, the low affinity target is selected from the group consisting of: NY-ESO-1, Sialyl Lewis X antigen and Tn antigen. In some embodiments, the low affinity target is a cell surface protein on an epithelial cancer cell. In some embodiments, the antibody target is a cell surface protein on a hematologic cancer cell. In some embodiments, the antibody target is selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, CD52 and CD70.

In some embodiments, a modified J-chain comprises a modified human J-chain sequence, or a functional fragment thereof. In some embodiments, the modified human J-chain sequence comprises the native human J-chain sequence of SEQ ID NO: 1. In some embodiments, the J-chain binding moiety is introduced into the native human J-chain sequence of SEQ ID NO: 1 by direct or indirect fusion. In some embodiments, the binding moiety is introduced by indirect fusion through a peptide linker. In some embodiments, the indirect fusion is through a peptide linker at or around a C- and/or an N-terminus of the binding moiety. In some embodiments, the binding moiety is introduced into the native human J-chain sequence of SEQ ID NO: 1 at or around the C-terminus. In some embodiments, the binding moiety is introduced into the native human J-chain sequence of SEQ ID NO: 1 within about 10 residues from the C-terminus. In some embodiments, the binding moiety is introduced into the native human J-chain sequence of SEQ ID NO: 1 at or around the N-terminus. In some embodiments, the binding moiety is introduced into the native human J-chain sequence of SEQ ID NO: 1 within about 10 amino acid residues from the N-terminus. In some embodiments, the binding moiety is introduced into the native human J-chain sequence in between cysteine residues 92 and 101 of SEQ ID NO: 1. In some embodiments, the binding moiety is introduced into the native human J-chain sequence of SEQ ID NO: 1 at or near a glycosylation site. In some embodiments, the peptide linker is about 10 to 20 amino acids long. In some embodiments, the peptide linker is about 15 to 20 amino acids long. In some embodiments, the peptide linker is 15 amino acids long.

In some embodiments, the binding moiety is introduced into the native human J-chain sequence of SEQ ID NO: 1 by chemical or chemo-enzymatic derivatization. In some embodiments, the binding moiety is introduced into the native human J-chain sequence of SEQ ID NO: 1 by a chemical linker. In some embodiments, the chemical linker is a cleavable or non-cleavable linker. In some embodiments, the cleavable linker is a chemically labile linker or an enzyme-labile linker. In some embodiments, the linker is selected from the group consisting of N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), iminothiolane (IT), bifunctional derivatives of imidoesters, active esters, aldehydes, bis-azido compounds, bis-diazonium derivatives, diisocyanates, and bis-active fluorine compounds. In some embodiments, the modified J-chain is modified by insertion of an enzyme recognition site, and by post-translationally attaching a binding moiety at the enzyme recognition site through a peptide or non-peptide linker.

In some embodiments, a binding moiety is selected from the group consisting of: antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, ligands and receptors. In some embodiments, the binding moiety is an antigen-binding fragment and is selected from the group consisting of: F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv, and single domain antibody. In some embodiments, the antigen-binding fragment is an scFv.

In some embodiments, the modified J-chain is in a V-linker-J orientation. In some embodiments, the modified J-chain is in a J-linker-V orientation. In some embodiments, the IgM, IgA, IgG/IgM or IgG/IgA antibody is a bispecific antibody. In some embodiments, the IgM, IgA, IgG/IgM or IgG/IgA antibody is a multispecific antibody.

Aspects of the invention include pharmaceutical compositions for the treatment of cancer, wherein the pharmaceutical composition comprises an effective amount of a binding molecule and a pharmaceutically acceptable carrier. Aspects of the invention include sse of a binding molecule in the preparation of a medicament for treating cancer. In some embodiments, the cancer is a hematologic cancer or an epithelial cancer. In some embodiments, the hematologic cancer is a leukemia, lymphoma, myeloma, or myelodysplastic syndrome. In some embodiments, the leukemia is an acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, or chronic lymphocytic leukemia. In some embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In some embodiments, the epithelial cancer is a melanoma, non-small-cell lung, nasopharyngeal, colorectal, liver, urinary bladder, ovarian, gastric, esophageal, pancreatic, renal, thyroid or breast cancer. In some embodiments, the breast cancer is hormone receptor negative or triple negative breast cancer.

In some embodiments, the medicament further comprises an effective amount of a second therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of an IgM pentamer, comprising a J-chain, wherein chains A and B are identical in native IgM.

FIG. 2 shows the schematic structures of IgA, dimeric IgA, and secretory IgA (sIgA).

FIG. 3 shows the amino acid sequence of mature human J-chain (SEQ ID NO: 1).

FIG. 4 is an illustration of the structure of Tn antigen.

FIG. 5 is a schematic illustration of two different orientations of J-chain constructs comprising a modified J-chain with a binding moiety that binds to CTLA4.

FIG. 6 is a schematic illustration of an asymmetric IgM pentamer with binding specificity for a target antigen and comprising a binding moiety attached to the J-chain.

FIG. 7 lists IgM, IgA, IgG/IgM or IgG/IgA antibody targets and targets for a binding moiety on a J-chain. Any of the antibody targets listed in the left column can be combined with any of the targets for a binding moiety on a J-chain listed in the right column.

FIG. 8 shows hybrid SDS PAGE and western blot analyses of various anti-PD-L1 IgM antibodies with an anti-CTLA4 binding moiety attached to the J-chain. Proper assembly of the pentameric PD-L1 binding IgM in the presence of a J-chain with or without anti-CTLA-4 scFv results in an increased amount of assembled product and a gel mobility shift.

FIG. 9, Panel A shows an SDS PAGE analysis of anti-CD20 IgM antibodies with or without various anti-CD3 binding moieties on the J-chain. Panel B is a graph showing results of a T-cell activation assay comparing the ability of an anti-CD20 IgM with a CD3-binding moiety on the J-chain to activate T-cells, as compared to anti-CD20 IgM antibodies without a CD3-binding moiety on the J-chain, as well as anti-CD20 IgG antibodies.

FIG. 10 shows two graphs that compare binding of anti-PD-L1 IgM and anti-PD-L1 IgG molecules in two different cell types having high and low levels of PD-L1 expression.

FIG. 11 shows a graph that compares inhibition of PD-1:PD-L1 interaction by IgM and IgG molecules made with VH sequences from anti-PD-L1 S70 antibody.

FIG. 12, Panel A shows an SDS PAGE hybrid gel of S70 IgM with the wild type or CD3-binding scFv fused J-chain. Panel B is a graph showing results from a PD-1:PD-L1 interaction blockade assay showing that the CD3-binding J chain does not disrupt PD-L1 binding and resultant blockade of activity.

FIG. 13 is a graph showing T-cell activation by S70 IgM carrying the wt or CD3-binding J-chain on a PD-L1 expressing cell line, with and without additional interferon gamma stimulation to increase PD-L1 expression.

FIG. 14 is a graph showing T-cell-dependent target cell killing with low (HDML2) and high (SUPHD) PD-L1 expressing cells.

FIG. 15, Panel A shows an SDS PAGE hybrid gel demonstrating expression and assembly of S70 IgM in the presence of wild type, CD3-binding (V) or anti-CTLA-4 (Y) scFv. Panel B is a graph that demonstrates blockade of PD-1:PD-L1 interaction with each of these antibodies.

FIG. 16, Panel A shows a hybrid gel of anti-PD-L1IgM with and without wt J-chain as well as with two modified J-chains carrying either an anti-CTLA-4 scFv (Y) or and anti-CD3 scFv(V).

FIG. 17 shows two graphs that show binding kinetics of S70 IgM carrying the CTLA-4 binding J-chain or the parent anti-CTLA-4 binding IgG, using Forte Bio BLI readout. The parent CTLA-4 binding antibody (IgG) binds with a Kd of 2 nM versus the monovalent binding of the S70 Y15J.

FIG. 18 is a schematic illustration comparing the ability of TRAIL ligand, IgG and IgM antibodies to target members of the tumor necrosis factor (TNF) superfamily.

FIG. 19 is a graph that compares the agonistic activity of anti-DR5 IgM and IgG antibodies.

 DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges encompassed within the invention, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provides one skilled in the art with a general guide to many of the terms used in the present application.

All publications mentioned herein are expressly incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

The term “antibody” includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab′)2, and Fv). The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Unless noted otherwise, the term “antibody” is used herein in the broadest sense and specifically includes all isotypes, sub-classes and forms of antibodies, including IgG, IgM, IgA, IgD, and IgE antibodies and their fragments, preferably antigen-binding fragments. Preferred antibodies herein include IgM and IgA antibodies and their antigen-binding fragments, which may be modified to include sequences from other isotypes, such as IgG to produce chimeric antibodies.

In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site.

IgM is a glycoprotein which forms polymers where multiple immunoglobulins are covalently linked together with disulfide bonds. IgM mostly exists as a pentamer but also as a hexamer and therefore contains 10 or 12 antigen binding sites. The pentameric form typically contains an additional polypeptide, called the J-chain, but can also be made in the absence of J-chain. The pentameric IgM molecule has a molecular weight of approximately 970 kDa. Due to its polymeric nature, IgM possesses high avidity and is particularly effective in complement activation. Unlike in IgG, the heavy chain in IgM monomers is composed of one variable and four constant domains. The IgM constant domains are designated herein as CM1 or Cμ1, CM2 or Cμ2, CM3 or Cμ3, and CM4 or Cμ4, wherein the “CM” and Cμ.” designations are used interchangeably. The structure of an IgM pentamer is illustrated in FIG. 1.

The term “IgM” is used herein in the broadest sense and specifically includes mono-, and multi-specific (including bispecific) IgM molecules, such as, for example, the multi-specific IgM binding molecules disclosed in PCT Application No. PCT/US2014/054079, the entire disclosure of which is expressly incorporated by reference herein.

The term “IgM binding unit” or “IgM antibody binding unit” is used in the broadest sense and specifically covers an IgM antibody heavy chain constant region polypeptide, comprising at least a CM4 constant domain, fused to a variable domain sequence (VH) binding to a target (e.g., antigen), with or without an associated antibody light chain variable domain (VL) sequence.

The term “bispecific IgM binding unit” or “bispecific IgM antibody binding unit” is used in the broadest sense and specifically covers a pair of IgM antibody heavy chain constant region polypeptides, comprising at least a CM4 constant domain, fused to a variable domain sequence (VH), each variable domain sequence binding to a different target, with or without associated antibody light chain variable domain (VL) sequences. In one embodiment, the bispecific IgM antibody comprises two VHVL antigen binding regions, each capable of binding to a different epitope on one antigen or epitopes on two different antigens. The bispecific IgM antibody binding units can be full length from a single species, or be chimerized or humanized. The bispecific IgM antibodies of the present invention have a penta- or hexameric ring structure comprising five or six bispecific IgM binding units.

The term “multi-specific IgM” is used herein in the broadest sense to refer to IgM antibodies with two or more binding specificities. Thus, the term “multi-specific” includes “bispecific”, e.g., bispecific antibodies or bispecific binding units, including IgM pentamers comprising at least two monospecific subunits, each binding to a different antigen (AA, BB), or five or six bispecific subunits, each binding to two different antigens (AB, AB). Thus, the bispecific and multi-specific IgM pentamers may include five identical bispecific binding units, monospecific IgM binding units, at least two of them have different binding specificities, or any combination thereof.

A “full length IgM antibody heavy chain” is a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain constant domain 1 (CM1 or Cμ1), an antibody heavy chain constant domain 2 (CM2 or Cμ2), an antibody heavy chain constant domain 3 (CM3 or Cμ3), and an antibody heavy chain constant domain 4 (CM4 or Cμ4). The bispecific full length IgM antibodies as defined herein comprise five or six monomers (binding units), each with two antigen binding sites, which specifically bind to two different binding targets (epitopes). The C-terminus of the heavy or light chain of the full length antibody denotes the last amino acid at the C-terminus of the heavy or light chain. The N-terminus of the heavy or light chain of the full length antibody denotes the first amino acid at the N-terminus of the heavy or light chain.

Native IgA is a tetrameric protein comprising two identical light chains (κ or λ) and two identical heavy chains (α). In the human, there are two IgA isotypes, IgA1 and IgA2. IgA, similarly to IgG, contains three constant domains (CA1-CA3 or Cα1-Cα3), with a hinge region between the Cα1 and Cα2 domains, wherein the “CA” and “Cα” designations are used interchangeably. All IgA isotypes have an 18 amino acid “tailpiece”, which is located C-terminal to the Cα3 domain, which enables polymeric Ig formation (see, e.g., Garcia-Pardo et al., 1981, J Biol. Chem. 256, 11734-11738 and Davis et al., 1988, Eur. J Immunol. 18, 1001-1008). Serum IgA is a monomer but can also polymerize. In its secretory form IgA comprises from 2-5 of the basic 4-chain units, linked by a J-chain, which may include a tail-piece, and may be associated by a secretory component. The structures of tail-piece, dimeric IgA and secretory IgA, associated with a secretory component (sIgA) are illustrated in FIG. 2. IgA antibodies can be further divided into IgA1 and IgA2 sub-classes. The term “IgA” antibody is used herein to specifically include all sub-classes, i.e., IgA1 and IgA2 antibodies, including dimeric and multimeric forms, with and without a secretory component, as well as fragments, preferably antigen-binding fragments, of such antibodies. For the purposes of the present invention, the IgA antibody preferably is a dimer, where two tail-pieces are connected by a J-chain (see, FIG. 2).

The term “IgA” is used herein in the broadest sense and specifically includes mono-, and multi-specific IgA molecules, such as, for example, the multi-specific IgA binding molecules disclosed in PCT Application No. PCT/US2015/015268, the entire disclosure of which is expressly incorporated by reference herein.

The term “multi-specific IgA” is used herein in the broadest sense to refer to IgA antibodies with two or more binding specificities. Thus, the term “multi-specific” includes “bispecific”, e.g., bispecific antibodies or bispecific binding units, including IgA dimers comprising two monospecific subunits, each binding to a different antigen (AA, BB), or two bispecific subunits, each binding to two different antigens (AB, AB).

In one embodiment, the dimeric multi-specific IgA molecules consist of two monospecific binding units, each binding unit having binding specificity to a different binding target (AA, BB). In another embodiment, in the dimeric IgA molecules at least one of the two binding units has two different binding specificities (i.e., is a bispecific, e.g., AA, A, B or AA, BC). In another embodiment, each of the two binding units has two specificities, which may be the same (AB, AB) or different (AC, CD or AB, AC, for example).

The term “bispecific IgA antibody binding unit” is used in the broadest sense and specifically covers a pair of IgA antibody heavy chain constant region polypeptides, comprising at least a CA3 constant domain, fused to a variable domain sequence (VH), each variable domain sequence binding to a different target, with or without associated antibody light chain variable domain (VL) sequences. In one embodiment, the bispecific IgA antibody comprises two VHVL antigen binding regions, each capable of binding to a different epitope on one antigen or epitopes on two different antigens. The bispecific IgA antibody binding units can be full length from a single species, or be chimerized or humanized.

A “full length IgA antibody heavy chain” is a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain constant domain 1 (CA1 or Cα1), an antibody constant heavy chain constant domain 2 (CA2 or Cα2), and an antibody heavy chain constant domain 3 (CA3 or Cα3). The bi- or multi-specific full length IgA antibodies according to the invention comprise two monomers (binding units), each of which may be mono- or bispecific, with or without a secretory component. Thus, the multi-specific IgA antibodies of the present invention may include monospecific and bispecific binding units, provided that the resultant IgA antibody has at least two binding specificities. The C-terminus of the heavy or light chain of the full length antibody denotes the last amino acid at the C-terminus of the heavy or light chain. The N-terminus of the heavy or light chain of the full length antibody denotes the first amino acid at the N-terminus of the heavy or light chain.

For further details of the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The term “interface”, as used herein, is used to refer to a region, which comprises those “contact” amino acid residues (or other non-amino acid groups such as, for example, carbohydrate groups) in a first IgM heavy chain constant region which interact with one or more “contact” amino acid residues (or other non-amino acid groups) in a second IgM heavy chain constant region.

The term “asymmetric interface” is used to refer to an interface (as hereinabove defined) formed between two antibody chains, such as a first and a second IgM heavy chain constant region and/or between an IgM heavy chain constant region and its matching light chain, wherein the contact residues in the first and the second chains are different by design, comprising complementary contact residues. The asymmetric interface can be created by knobs/holes interactions and/or salt bridges coupling (charge swaps) and/or other techniques known in the art, such as for example, by the CrossMab approach for coupling a μ heavy chain to its matching light chain.

A “cavity” or “hole” refers to at least one amino acid side chain which is recessed from the interface of the second polypeptide and therefore accommodates a corresponding protuberance (“knob”) on the adjacent interface of the first polypeptide. The cavity (hole) may exist in the original interface or may be introduced synthetically (e.g., by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one “import” amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the second polypeptide. The preferred import residues for the formation of a cavity are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T), valine (V) and glycine (G). Most preferred amino acid residues are serine, alanine or threonine, most preferably alanine. In the preferred embodiment, the original residue for the formation of the protuberance has a large side chain volume, such as tyrosine (Y), arginine (R), phenylalanine (F) or tryptophan (W).

An “original” amino acid residue is one which is replaced by an “import” residue which can have a smaller or larger side chain volume than the original residue. The import amino acid residue can be a naturally occurring or non-naturally occurring amino acid residue, but preferably is the former.

By “non-naturally occurring” amino acid residue is meant a residue which is not encoded by the genetic code, but which is able to covalently bind adjacent amino acid residue(s) in the polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991), for example. To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244: 182 (1989) and Ellman et al., supra can be used. Briefly, this involves chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA. The methods of the current invention, in certain embodiments, involve replacing at least one original amino acid residue in an IgM heavy chain, but more than one original residue can be replaced. Normally, no more than the total residues in the interface of the first or second polypeptide will comprise original amino acid residues which are replaced. The preferred original residues for replacement are “buried”. By “buried” is meant that the residue is essentially inaccessible to solvent. The preferred import residue is not cysteine to prevent possible oxidation or mispairing of disulfide bonds.

The protuberance is “positionable” in the cavity which means that the spatial location of the protuberance and cavity on the interface of the first polypeptide and second polypeptide respectively and the sizes of the protuberance and cavity are such that the protuberance can be located in the cavity without significantly perturbing the normal association of the first and second polypeptides at the interface. Since protuberances such as Tyr, Phe and Trp do not typically extend perpendicularly from the axis of the interface and have preferred conformations, the alignment of a protuberance with a corresponding cavity relies on modeling the protuberance/cavity pair based upon a three-dimensional structure such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using widely accepted techniques in the art, including techniques of molecular modeling.

By “original nucleic acid” is meant the nucleic acid encoding a polypeptide of interest which can be “altered” (i.e., genetically engineered or mutated) to encode a protuberance or cavity. The original or starting nucleic acid may be a naturally occurring nucleic acid or may comprise a nucleic acid which has been subjected to prior alteration (e.g., a humanized antibody fragment). By “altering” the nucleic acid is meant that the original nucleic acid is mutated by inserting, deleting or replacing at least one codon encoding an amino acid residue of interest. Normally, a codon encoding an original residue is replaced by a codon encoding an import residue. Techniques for genetically modifying a DNA in this manner have been reviewed in Mutagenesis: a Practical Approach, M. J. McPherson, Ed., (IRL Press, Oxford, UK. (1991), and include site-directed mutagenesis, cassette mutagenesis and polymerase chain reaction (PCR) mutagenesis, for example.

The protuberance or cavity can be “introduced” into the interface of the first or second polypeptide by synthetic means, e.g., by recombinant techniques, in vitro peptide synthesis, those techniques for introducing non-naturally occurring amino acid residues previously described, by enzymatic or chemical coupling of peptides or some combination of these techniques. According, the protuberance or cavity which is “introduced” is “non-naturally occurring” or “non-native”, which means that it does not exist in nature or in the original polypeptide (e.g., a humanized monoclonal antibody).

Preferably the import amino acid residue for forming the protuberance has a relatively small number of “rotamers” (e.g., about 3-6). A “rotamer” is an energetically favorable conformation of an amino acid side chain. The number of rotamers for the various amino acid residues are reviewed in Ponders and Richards, J. Mol. Biol. 193: 775-791 (1987).

Unless stated otherwise, the term “antibody” specifically includes native human and non-human IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD and IgM antibodies, including naturally occurring variants. Thus, for example, the human IgM sequence is given as SEQ ID NO: 110, while variants have been reported as SEQ ID NOs: 111-115.

The term “native” with reference to a polypeptide (e.g., an antibody or a J-chain) is used herein to refer to a polypeptide having a sequence that occurs in nature, regardless of its mode of preparation. Thus, the terms “native” and “native sequence” are used herein interchangeably, and expressly encompass recombinant polypeptides with a sequence that is found in nature.

The term “native sequence J-chain” or “native J-chain” as used herein refers to J-chain of native sequence IgM or IgA antibodies of any animal species, including mature human J-chain, the amino acid sequence of which is shown in FIG. 3 (SEQ ID NO: 1).

The term “modified J-chain” is used herein to refer to variants of native sequence J-chain polypeptides comprising an extraneous binding moiety introduced into the native sequence. The introduction can be achieved by any means, including direct or indirect fusion of an extraneous binding moiety or by attachment through a chemical linker. The term “modified human J-chain” specifically encompasses, without limitation, a native sequence human J-chain of the amino acid sequence of SEQ ID NO: 1 modified by the introduction of a binding moiety. The term specifically encompasses, without limitation, a native sequence human J-chain of the amino acid sequence of SEQ ID NO: 1 modified by the introduction of an extraneous binding moiety which does not interfere with efficient polymerization (dimerization) of IgM or IgA and binding of such polymers (dimers) to a target

The term “binding moiety” is used herein in the broadest sense to encompass any chemical entity capable of specific binding to a target, such as an antigen. Examples of binding moieties include, without limitation, antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, ligands and receptors. Preferred binding moieties are polypeptides (including peptides), preferably with a biological function. An example of a biological function is the ability of a binding moiety to bind to and activate or block the activity of a signaling pathway.

The term “polypeptide” is used herein in the broadest sense and includes peptide sequences. The term “peptide” generally describes linear molecular chains of amino acids containing up to about 60, preferably up to about 30 amino acids covalently linked by peptide bonds.

The term “extraneous” with reference to a “binding moiety” is used herein to refer to a binding moiety not present in a reference native polypeptide sequence at the same location. Thus, an extraneous polypeptide sequence (including peptide sequences), might be comprised within the corresponding native sequence but at a different location. In a preferred embodiment, the “extraneous” sequence is not present in the corresponding native sequence in any location.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597, for example.

The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

“Humanized” forms of non-human (e.g., murine) antibodies are antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are also replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

An “isolated” antibody herein is one which has been identified and separated and/or recovered from a component of its natural environment in a recombinant host cell. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes, as well as undesired byproducts of the production. In a preferred embodiment, an isolated antibody herein will be purified (1) to greater than 95% by weight, or greater than 98% by weight, or greater than 99% by weight, as determined by SDS-PAGE or SEC-HPLC methods, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a amino acid sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, an isolated antibody will be prepare by at least one purification step.

The term “specific binding” or “specifically binds to” or is “specific for” refers to the binding of a binding moiety to a binding target, such as the binding of an antibody to a target antigen, e.g., an epitope on a particular polypeptide, peptide, or other target (e.g., a glycoprotein target), and means binding that is measurably different from a non-specific interaction (e.g., a non-specific interaction may be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining binding of a binding moiety, or an antibody, or an antibody modified by introduction of a binding moiety, to a target molecule compared to binding to a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or greater. In certain instances, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

“Binding affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). For example, the Kd can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or stronger. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art.

As used herein, the “Kd” or “Kd value” refers to a dissociation constant measured by a technique appropriate for the antibody and target pair, for example using surface plasmon resonance assays, for example, using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at about 10 response units (RU).

The terms “conjugate,” “conjugated,” and “conjugation” refer to any and all forms of covalent or non-covalent linkage, and include, without limitation, direct genetic or chemical fusion, coupling through a linker or a cross-linking agent, and non-covalent association.

The term “fusion” is used herein to refer to the combination of amino acid sequences of different origin in one polypeptide chain by in-frame combination of their coding nucleotide sequences. The term “fusion” explicitly encompasses internal fusions, i.e., insertion of sequences of different origin within a polypeptide chain, in addition to fusion to one of its termini. The term “fusion” is used herein to refer to the combination of amino acid sequences of different origin

The term “valent” as used herein denotes the presence of a specified number of binding sites in an antibody. As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively. Thus, if in a bispecific IgA antibody according to the present invention each binding unit is bivalent, the bispecific IgA antibody will have 4 valencies.

The term “epitope” includes any molecular determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. A “binding region” is a region on a binding target bound by a binding molecule.

“Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). “Monospecific” refers to the ability to bind only one epitope. According to one embodiment the bispecific IgM antibody binds to each epitope with an affinity of at least 10−7M, or 10−8 M or better.

The term “target” or “binding target” is used in the broadest sense and specifically includes polypeptides, without limitation, nucleic acids, carbohydrates, lipids, cells, and other molecules with or without biological function as they exist in nature.

The term “antigen” refers to an entity or fragment thereof, which can bind to an antibody or trigger a cellular immune response. An immunogen refers to an antigen, which can elicit an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term antigen includes regions known as antigenic determinants or epitopes, as defined above.

As used herein, the term “immunogenic” refers to substances, which elicit the production of antibodies, and/or activate T-cells and/or other reactive immune cells directed against an antigen of the immunogen.

An “antigen-binding site” or “antigen-binding region” of an antibody of the present invention typically contains six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences and/or structural information from antibody/antigen complexes. Also included within the scope of the invention are functional antigen binding sites comprised of fewer CDRs (i.e., where binding specificity is determined by three, four or five CDRs). Less than a complete set of 6 CDRs may be sufficient for binding to some binding targets. Thus, in some instances, the CDRs of a VH or a VL domain alone will be sufficient. Furthermore, certain antibodies might have non-CDR-associated binding sites for an antigen. Such binding sites are specifically included within the present definition.

The term “host cell” as used in the current application denotes any kind of cellular system which can be engineered to generate the antibodies according to the current invention. In one embodiment Chinese hamster ovary (CHO) cells are used as host cells.

As used herein, the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

A nucleic acid is “operably linked” when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The term “antagonist” as used herein refers to a molecule that causes a decrease in a function or activity as compared to the same function or activity in the absence of the molecule. An “antagonist” of a signaling pathway is therefore a molecule whose presence causes a decrease in a function or activity of the signaling pathway. The term “antagonize” as used herein refers to causing a decrease in a function or activity.

The term “agonist” as used herein refers to a molecule that causes an increase in a function or activity as compared to the same function or activity in the absence of the molecule. An “agonist” of a signaling pathway is therefore a molecule whose presence causes an increase in a function or activity of the signaling pathway. The term “agonize” as used herein refers to causing an increase in a function or activity.

The term “T-cell inhibitory signaling pathway” as used herein refers to a T-cell signaling pathway that leads to a qualitative or quantitative decrease in, blocking or, or halting of a T-cell immune response.

The term “T-cell stimulatory signaling pathway” as used herein refers to a T-cell signaling pathway that leads to a qualitative or quantitative increase in or maintenance of a T-cell immune response.

The term “low level expression target” as used herein refers to a target whose expression level on a target cell ranges from 0 to 1+, as determined by immunohistochemistry (IHC) tissue analysis, preferably performed on frozen, formalin-fixed, paraffin-embedded tissue sections. Guidelines for determining expression level via IHC are provided, for example, by the College of American Pathologists (CAP), and are exemplified by the ASCO-CAP HER2 Test Guideline Recommendations, available on the World Wide Web at cap.org/apps/docs/committees/immunohistochemistry/summary_of_recommendations.pdf.

The term “low affinity target” as used herein refers to a target whose binding interaction with an antibody has a dissociation constant Kd that is greater than or equal to a value ranging from about 10 to 100 nM, such as about 25 to about 75 nM, as measured by ELISA.

Design and Production of Binding Molecules with Modified J-Chain

IgM is the first immunoglobulin produced by B cells in response to stimulation by antigen, and is present at around 1.5 mg/ml in serum with a half-life of 5 days. IgM is a pentameric or hexameric molecule. Just as IgG, IgM monomers consist of two light and two heavy chains. However, while IgG contains three heavy chain constant domains (CH1, CH2 and CH3), the heavy (μ) chain of IgM additionally contains a fourth constant domain (CH4), similarly to the ε heavy chains in IgE. This extra constant domain is located in place of the IgG and IgA proline-rich hinge region that is responsible for the rotational flexibility of the antigen-binding Fab domains relative to the Fc domain of IgG and IgA antibodies.

Five IgM monomers form a complex with an additional small polypeptide chain (the J-chain) to form a native IgM molecule. The J-chain is considered to facilitate polymerization of μ chains before IgM is secreted from antibody-producing cells. While crystallization of IgM has proved to be notoriously challenging, Czajkowsky and Shao (PNAS 106(35):14960-14965, 2009) recently published a homology-based structural model of IgM, based on the structure of the IgE Fc domain and the known disulfide pairings. The authors report that the human IgM pentamer is a mushroom-shaped molecule with a flexural bias. The IgM heavy (p) chain contains five N-linked glycosylation sites: Asn-171, Asn-332, Asn-395, Asn-402 and Asn-563.

Immunoglobulin A (IgA), as the major class of antibody present in the mucosal secretions of most mammals, represents a key first line of defense against invasion by inhaled and ingested pathogens. IgA is also found at significant concentrations in the serum of many species, where it functions as a second line of defense mediating elimination of pathogens that have breached the mucosal surface. Receptors specific for the Fc region of IgA, FcαR, are key mediators of IgA effector function. Human IgA may have two different IgA heavy constant region (Ca) genes which give rise to the two subclasses, IgA1 and IgA2. The main difference between IgA1 and IgA2 resides in the hinge region that lies between the two Fab arms and the Fc region. IgA1 has an extended hinge region due to the insertion of a duplicated stretch of amino acids, which is absent in IgA2. IgA has the capacity to form dimers, in which two monomer units, each comprising two heavy chains and light chains, are postulated to be arranged in an end-to-end configuration stabilized by disulfide bridges and incorporation of a J-chain. Dimeric IgA, produced locally at mucosal sites, is transported across the epithelial cell boundary and out into the secretions by interaction with the polymeric immunoglobulin receptor (pIgR). During this process the pIgR is cleaved and the major fragment, termed secretory component (SC), becomes covalently attached to the IgA dimer.

Both IgA and IgM possess an 18-amino acid extension in the C terminus called the “tail-piece” (tp). The IgM (μtp) and IgA (αtp) tail-pieces differ at seven amino acid positions. The IgM and IgA tail-piece is highly conserved among various animal species. The conserved penultimate cysteine residue in the IgA and IgM tail-pieces has been demonstrated to be involved in polymerization. Both tail-pieces contain an N-linked carbohydrate addition site, the presence of which is required for dimer formation in IgA and J-chain incorporation and pentamer formation in IgM. However, the structure and composition of the N-linked carbohydrates in the tail-pieces differ, suggesting differences in the accessibility of the glycans to processing by glycosyltransferases.

The nucleotide and/or protein sequences of J-chains of human, and various vertebrate animal species, such as cow, mouse, avian, amphibian, and rabbit, have been reported. The human J-chain contains eight cysteine residues, two (Cys13 and Cys69) are involved in disulfide bridges with the α or μ-chains (in IgA and IgM, respectively), and six are involved in intrachain disulfide bridges (Cys13: Cys101, Cys72: Cys92, Cys109: Cys134). The three-dimensional crystal structure of the J-chain has not been reported.

The binding molecules of the present invention include a J-chain that comprises a binding moiety that antagonizes a T-cell inhibitory signaling pathway, without interfering with the ability of the IgM, IgA, IgG/IgM or IgG/IgA antibody to bind to its binding target(s). A binding molecule can, for example, be an IgM antibody, an IgA antibody, or an IgG/IgM or IgG/IgA hybrid antibody, which may contain an IgM or IgA tail-piece at the IgG heavy chain and thus combine the properties of IgG and IgA or IgA, including the ability to incorporate and form polymers with a modified J-chain whose binding moiety antagonizes a T-cell inhibitory signaling pathway. For further details on IgG/IgM and IgG/IgA hybrid antibodies see, e.g., Koteswara et al., Clinical Immunology 2001, 101(1):21-31. An illustration of an example binding molecule in accordance with aspects of the invention is depicted in FIG. 6. The depicted binding molecule comprises an IgM pentamer with binding specificity for a target antigen, and comprises a binding moiety attached to the J-chain.

T-cell inhibitory signaling pathways are known in the art, and include, without limitation, those described in Pardoll, Drew M. “The blockade of immune checkpoints in cancer immunotherapy.” Nature Reviews Cancer 12.4 (2012): 252-264, the disclosure of which is herein incorporated by reference in its entirety. Non-limiting examples of T-cell inhibitory signaling pathways and components thereof are described in further detail below.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is a member of the immunoglobulin superfamily and has been shown to transmit an inhibitory signal to T-cells. The membrane-bound isoform of CTLA4 functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. E.g., Pardoll at 255.

In addition to CTLA4, other T-cell inhibitory signaling pathways include, for example, those involving programmed cell death-1 (PD-1) and its ligand, programmed cell death ligand-1 (PD-L1). PD-1 is an inhibitory cell surface receptor protein of the immunoglobulin superfamily, and is involved in the regulation of T-cell function in immunity and self-tolerance. PD-L1 interacts with PD-1 on the surface of T-cells, and inhibits proliferation of T-cells by blocking cell cycle progression and cytokine production. Id.

Another example of a T-cell inhibitory signaling pathway is the signaling pathway involving T-cell immunoglobulin and mucin domain 3 (TIM3). TIM3 is a cell surface glycoprotein that is expressed on the surface of T-cells, and functions as an inhibitory molecule that is involved in the termination of Th1 cells. Id.

Another example of a T-cell inhibitory signaling pathway is the signaling pathway involving lymphocyte-activation gene 3 (LAG3). LAG3 belongs to the immunoglobulin superfamily, and functions as an inhibitor of cellular proliferation, activation and homeostasis of T-cells. Id.

Another example of a T-cell inhibitory signaling pathway is the signaling pathway involving B- and T-lymphocyte attenuator protein (BTLA). BTLA is a cell surface protein that functions by inhibiting T-cells via interaction with members of the tumor necrosis factor receptor superfamily. BTLA is known to negatively regulate T-cell immune responses. Id.

Another example of a T-cell inhibitory signaling pathway is the signaling pathway involving V-domain Ig suppressor of T-cell activation (VISTA). VISTA is a regulator of T-cell function that is expressed on hematopoietic cells and leukocytes, and functions by suppressing T-cell activation. E.g., Lines J L, et al., Cancer research. 2014; 74(7):1924-1932.

Another example of a T-cell inhibitory signaling pathway is the signaling pathway involving the protein T-cell immunoreceptor with Ig and ITIM Domains (TIGIT). TIGIT is expressed in several classes of T-cells, and binds with high affinity to the poliovirus receptor. TIGIT suppresses T-cell activation by promoting generation of mature immunoregulatory dendritic cells. E.g., Yu X et al., Nat Immunol. 2009 January; 10(1):48-57.

As reviewed above, the subject binding molecules comprise a binding moiety on the J-chain that antagonizes a T-cell inhibitory signaling pathway. In some embodiments, a binding moiety on the J-chain binds to a target in a T-cell inhibitory signaling pathway, and thereby blocks or diminishes inhibitory signals that are received by a T-cell via the pathway. As a result, the T-cell's immune response is not blocked, halted or diminished, or, at least, the inhibition of the T-cell's immune response is reduced or diminished. The binding moiety on the J-chain of a subject binding molecule can be used to antagonize any T-cell inhibitory signaling pathway, including but not limited to the inhibitory signaling pathways that involve the proteins listed in Table 1, below. The GenBank Accession Numbers corresponding to the human protein sequences of these T-cell inhibitory signaling pathway targets are provided in Table 1, below.

TABLE 1 Sequence information for T-cell inhibitory signaling pathway targets T-cell inhibitory signaling SEQ ID pathway member: NO. CTLA4 16 PD-1 17 TIM3 18 LAG3 19 BTLA 20 VISTA 21 TIGIT 22

A binding moiety on the J-chain of a subject binding molecule can include, without limitation, antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antigen-binding fragments of antibody-drug conjugate, antibody-like molecules, antigen-binding fragments of antibody-like molecules, soluble and membrane-bound proteins, ligands and receptors. It is emphasized that any type of binding moiety can be introduced into a J-chain, following the teaching of the present disclosure, by appropriately selecting the location and type of addition (e.g., direct or indirect fusion, chemical tethering, etc.).

In a preferred embodiment, a binding moiety on a J-chain is an antibody or an antigen-binding fragment of an antibody (also referred to as an “antibody fragment”), including monospecific, bispecific, and multi-specific antibodies and antibody fragments, that functions as an antagonist of a T-cell inhibitory signaling pathway. The term “antibody fragment” is used in the broadest sense and includes, without limitation, Fab, Fab′, F(ab′)2, scFv, and (scFv)2 fragments, complementarity determining region (CDR) fragments, linear antibodies, single-chain antibody molecules, minibodies, and multi-specific antibodies formed from antibody fragments. In a preferred embodiment, the antibody fragment is a single chain Fv (scFv).

In another preferred embodiment, a binding moiety on a J-chain is an antibody-like molecule, such as, for example, a human domain antibody (dAb), Dual-Affinity Re-Targeting (DART) molecule, a diabody, a di-diabody, dual-variable domain antibody, a Stacked Variable Domain antibody, a Small Modular ImmunoPharmaceutical (SMIP), a Surrobody, a strand-exchange engineered domain (SEED)-body, or TandAb that functions as an antagonist of a T-cell inhibitory signaling pathway.

A binding moiety on a J-chain can be introduced into a native J-chain sequence at any location that allows the binding of the binding moiety to its binding target without interfering with the binding of the recipient IgM, IgA, IgG/IgM or IgG/IgA molecule to its binding target or binding targets. Preferred locations include at or near the C-terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J-chain is accessible. In preferred embodiments, the binding moiety is introduced into the native sequence J-chain without about 10 residues from the C-terminus or without about 10 amino acid residues from the N-terminus, where the native sequence J-chain preferably is human J-chain of SEQ ID NO: 1. In another embodiment, the binding moiety is introduced into the native sequence human J-chain of SEQ ID NO: 1 in between cysteine residues 92 and 101 of SEQ ID NO: 1, or at an equivalent location of another native sequence J-chain. In a further embodiment, the binding moiety is introduced into a native sequence J-chain, such as a J-chain of SEQ ID NO: 1, at or near a glycosylation site. Most preferably, the binding moiety is introduced into the native sequence human J-chain of SEQ ID NO: 1 within about 10 amino acid residues from the C-terminus.

Introduction can be accomplished by direct or indirect fusion, i.e., by the combination of the J-chain and binding moiety amino acid sequences in one polypeptide chain by in-frame combination of their coding nucleotide sequences, with or without a peptide linker. The peptide linker (indirect fusion), if used, may, for example, be about 1 to 50, or about 1 to 40, or about 1 to 30, or about 1 to 20, or about 1 to 10, or about 10 to 20 amino acid residues, and may be present at one or both ends of the binding moiety to be introduced into the J-chain sequence. In a preferred embodiment, the peptide linker is about 10 to 20, or 10 to 15 amino acids long. In another preferred embodiment, the peptide linker is 15 amino acids long.

A J-chain binding moiety can also be appended to a native J-chain sequence by chemical linkage using heterobifunctional protein crosslinkers containing two different functional groups, which have their own reactivity and selectivity. These crosslinkers can be used in a one step process or can be used to create activated proteins, which can often be preserved and reacted with the second biomolecule in a separate step. Thus, for example, a heterobifunctional crosslinking reagent can be used to form conjugates between a J-chain and a binding moiety. The reactive groups include, without limitation imine reactive groups (such as NHS or Sulfo-NHS), maleimide groups, and the like. Such crosslinkers, which can be cleavable or non-cleavable, have been used, for example, in the formation of hapten carrier proteins and in preparing enzyme-antibody conjugates. Chemically, the cleavable crosslinkers specifically include, without limitation, disulfide-based, hydrazone, and peptide linkers. A well-known and much studied enzyme-labile linker is a valine-citrulline linker but other peptide linkers are also known and suitable. Typical representatives of non-cleavable linkers include thioethers, such as SMCC (N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate). For further details see, e.g., Ducry L and Stump B, Bioconjugate Chem. 2010, 21:5-13, the entire disclosure of which is expressly incorporated by reference herein. For listing of further suitable linkers see, e.g., Klein et al., Protein Engineering, Design & Selection; 2014, 27(10): 325-330, the entire disclosure of which is expressly incorporated by reference herein.

In some embodiments, a binding molecule comprises an amino acid sequence listed in Table 7. In some embodiments, a binding molecule comprises an amino acid sequence that is substantially similar to an amino acid sequence listed in Table 7, for example, has at least about 80% amino acid sequence identity, alternatively, has about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. 99.5%, or about 99.9% amino acid sequence identity to an amino acid sequence that is listed in Table 7.

While the modified J-chain usually contains one extraneous binding moiety, it is also possible to introduce more than one binding moiety into a J-chain. In some embodiments, a modified J-chain comprises one extraneous binding moiety. In some embodiments, a modified J-chain comprises more than one extraneous binding moiety. For example, in some embodiments, one binding moiety is introduced into a modified J-chain at either the N-terminus or the C-terminus. In some embodiments, a first binding moiety is introduced into a modified J-chain at the N-terminus, and a second binding moiety is introduced into the same modified J-chain at the C-terminus. For examples, in some embodiments, a first binding moiety is introduced into a modified J-chain at the N-terminus, and a second binding moiety is introduced into the same modified J-chain at the C-terminus. A binding molecule that comprises a binding moiety at both the N-terminus and the C-terminus of the J-chain is referred to herein as a binding molecule that comprises a “bidentate” J-chain.

The modified J-chain may be produced by well-known techniques of recombinant DNA technology, by expressing nucleic acid encoding the modified J-chain in a suitable prokaryotic or eukaryotic host organism, such as CHO cells or E. coli. Thus, the modified J-chain may, for example, be expressed in E. coli, as described by Symersky et al., Mol Immunol 2000, 37:133-140.

In one embodiment, the J-chain can be initially modified by insertion of an enzyme recognition site, and post-translationally modified by a peptide or non-peptide linker, which can tether any extraneous binding moiety to the J-chain, such as, for example, cytotoxic small molecule to make an antibody-drug conjugate (ADC).

The modified J-chain can also be co-expressed with the heavy and light chains of the recipient IgM, IgA, IgG/IgM or IgG/IgA antibody. Although due to its complex structure, the large scale production of recombinant IgM has been difficult, several recombinant production systems for IgM using non-lymphoid cells have been reported, including co-expression of the IgM heavy (H) and light (L) chains in C6 glioma cells, CHO cells, and HeLa cells (see, e.g. WO89/01975 and Wood et al., J. Immunol. 145, 3011-3016 (1990) for expression in CHO cells). Expression of an IgM monoclonal antibody in E. coli, with or without a J-chain, is described, e.g., in Azuma et al., Clin Cancer Res 2007, 13(9):2745-2750. Production of IgM in an immortalized human retina cell line expressing E1A and E1B proteins of an adenovirus is described in U. S. Application Publication No. 20060063234.

The recipient IgM, IgA, IgG/IgM or IgG/IgA antibody may be monospecific, bispecific or multi-specific. Bispecific and multi-specific IgM and IgA binding molecules, including antibodies, are described, for example, in PCT Application No. PCT/US2014/054079 and PCT/US2015/015268, the entire disclosures of which are expressly incorporated by reference herein.

A subject binding molecule can bind to any binding target via the IgM, IgA, IgG/IgM or IgG/IgA antibody, while the J-chain binding moiety antagonizes a T-cell inhibitory signaling pathway. As such, the subject binding molecules can be used to localize the functionality of the J-chain binding moiety to the location of a binding target that is targeted by the IgM, IgA, IgG/IgM or IgG/IgA antibody. Classes of antibody targets are described in further detail below.

Antagonist Targets

Aspects of the invention include binding molecules having an IgM, IgA, IgG/IgM or IgG/IgA antibody that antagonizes a T-cell inhibitory signaling pathway. T-cell inhibitory signaling pathways are known in the art, and include, without limitation, those described in Pardoll et al. Non-limiting examples of T-cell inhibitory signaling pathways and components thereof are described in further detail below.

One example of a T-cell inhibitory signaling pathway is the signaling pathway involving programmed cell death-1 (PD-1) and its ligand, programmed cell death ligand-1 (PD-L1). PD-1 is an inhibitory cell surface receptor protein of the immunoglobulin superfamily, and is involved in the regulation of T-cell function in immunity and self-tolerance. PD-L1 interacts with PD-1 on the surface of T-cells, and inhibits proliferation of T-cells by blocking cell cycle progression and cytokine production. Id.

Another example of a T-cell inhibitory signaling pathway is the signaling pathway involving T-cell immunoglobulin and mucin domain 3 (TIM3). TIM3 is a cell surface glycoprotein that is expressed on the surface of T-cells, and functions as an inhibitory molecule that is involved in the termination of Th1 cells. Id.

Another example of a T-cell inhibitory signaling pathway is the signaling pathway involving lymphocyte-activation gene 3 (LAG3). LAG3 belongs to the immunoglobulin superfamily, and functions as an inhibitor of cellular proliferation, activation and homeostasis of T-cells. Id.

As reviewed above, the subject binding molecules comprise a J-chain binding moiety that antagonizes a T-cell inhibitory signaling pathway. In some embodiments, an IgM, IgA, IgG/IgM or IgG/IgA antibody binds to a target that is involved in a T-cell inhibitory signaling pathway and antagonizes the inhibitory signaling pathway, thereby blocking or diminishing inhibitory signals that are received by a T-cell via the pathway, while the J-chain binding moiety also antagonizes a T-cell inhibitory signaling pathway. Due to their higher avidity, the subject IgM, IgA, IgG/IgM or IgG/IgA antibodies act more effectively as antagonists when directed against T-cell inhibitory signaling pathway targets, as compared to IgG antibodies, which only have two binding sites. As a result, the T-cell's immune response is not blocked, halted or diminished, or, at least, the inhibition of the T-cell's immune response is reduced or diminished. The antibody of a subject binding molecule can be used to antagonize any T-cell inhibitory signaling pathway, including but not limited to the inhibitory signaling pathways that involve the proteins listed in Table 2, below. The GenBank Accession Numbers corresponding to the human protein sequences of these T-cell inhibitory signaling pathway targets are provided in Table 2, below.

TABLE 2 Sequence information for T-cell stimulatory signaling pathway targets T-cell stimulatory signaling SEQ ID pathway member: NO. PD-1 23 PD-Ll 24 TIM3 25 LAG3 26

Agonist Targets

Aspects of the invention include binding molecules having an IgM, IgA, IgG/IgM or IgG/IgA antibody that agonizes a T-cell stimulatory signaling pathway. T-cell stimulatory signaling pathways are known in the art, and include, without limitation, those described in Pardoll et al. Non-limiting examples of T-cell stimulatory signaling pathways and components thereof are described in further detail below.

CD137 is a member of the tumor necrosis factor receptor (TNF-R) superfamily, and is expressed on the surface of T-cells. Its function is to stimulate T-cell proliferation and cytokine secretion. E.g., Pardoll at 254. OX40 is another member of the tumor necrosis factor receptor superfamily that is expressed on T-cells, and it functions by delivering a stimulatory signal to T-cells that helps to maintain the immune response over time. Id.

Another T-cell stimulatory signaling pathway involves CD40. CD40 is a member of the tumor necrosis factor receptor superfamily, and is expressed on antigen presenting cells. Engagement of CD40 with its ligand CD40L results in various T-cell stimulatory signals. Id.

Another T-cell stimulatory signaling pathway involves gluococorticoid-induced TNFR-related protein (GITR). GITR is a member of the tumor necrosis factor receptor superfamily, and is expressed on T-cells. It functions by increasing T-cell proliferation, activation and cytokine production. E.g., Nocentini, G. et al., Proc Natl Acad Sci USA. 1997 Jun. 10; 94(12): 6216-21.

CD27 is another protein that is involved in a T-cell stimulatory signaling pathway. Another member of the tumor necrosis factor receptor superfamily, CD27 is expressed on the surface of T-cells and functions by delivering a stimulatory signal to T-cells when it interacts with CD70. E.g., Pardoll at 254.

Another T-cell stimulatory signaling pathway involves herpesvirus entry mediator (HVEM). HVEM is a member of the tumor necrosis factor receptor superfamily, and is expressed on the surface of antigen presenting cells. When HVEM interacts with certain ligands, such as CD258, it delivers a stimulatory signal to T-cells. Id.

As reviewed above, the subject binding molecules comprise a binding moiety on the J-chain that antagonizes a T-cell inhibitory signaling pathway. In some embodiments, an IgM, IgA, IgG/IgM or IgG/IgA antibody binds to a target that is involved in a T-cell stimulatory signaling pathway and agonizes the stimulatory signaling pathway, thereby maintaining or increasing stimulatory signals that are received by a T-cell via the pathway, while the binding moiety on the J-chain antagonizes a T-cell inhibitory signaling pathway. Due to their higher avidity, the subject IgM, IgA, IgG/IgM or IgG/IgA antibodies act more effectively as agonists when directed against T-cell stimulatory signaling pathway targets, as compared to IgG antibodies, which only have two binding sites. As a result, a T-cell's immune response is maintained or increased. An antibody of a subject binding molecule can be used to agonize any T-cell stimulatory signaling pathway, including but not limited to the stimulatory signaling pathways that involve the proteins listed in Table 3, below. The GenBank Accession Numbers corresponding to the human protein sequences of these T-cell stimulatory signaling pathway targets are provided in Table 3, below.

TABLE 3 Sequence information for T-cell stimulatory signaling pathway targets T-cell stimulatory signaling SEQ ID pathway member: NO. CD137 (4-1BB) 116 OX40 117 CD40 118 GITR 119 CD27 120 HVEM 121

Other non-limiting examples of T-cell stimulatory signaling pathways include those mediated by: TNFR1 (DR1) (SEQ ID NO: 122); TNFR2 (SEQ ID NO: 123); Fas (CD95, Apo1, DR2) (SEQ ID NO: 124); CD30 (SEQ ID NO: 125); TRAILR1 (DR4, Apo2) (SEQ ID NO: 126); DR5 (TRAILR2) (SEQ ID NO: 127); TRAILR3 (DcR1) (SEQ ID NO: 128); TRAILR4 (DcR2) (SEQ ID NO: 129); OPG (OCIF) (SEQ ID NO: 130); TWEAKR (FN14) (SEQ ID NO: 131); DcR3 (SEQ ID NO: 132); DR3 (SEQ ID NO: 133); EDAR (SEQ ID NO: 134); and XEDAR (SEQ ID NO: 135). See, e.g., Aggarwal et al., Blood, 119:651-665, 2012, the disclosure of which is herein incorporated by reference in its entirety. In some embodiments, an IgM, IgA, IgG/IgM or IgG/IgA antibody binds to any one of these targets and agonizes a T-cell stimulatory signaling pathway, thereby maintaining or increasing stimulatory signals that are received by a T-cell via the pathway, while the binding moiety on the J-chain antagonizes a T-cell inhibitory signaling pathway.

Low Level Expression Targets

Aspects of the invention include binding molecules having an IgM, IgA, IgG/IgM or IgG/IgA antibody that binds to a low level expression target. Due to their higher avidity, the subject binding molecules are more potent than IgG antibodies. As such, the subject binding molecules can be employed in settings where a particular binding target is expressed at a low level, and where higher avidity is beneficial in facilitating binding between an antibody and a target. An antibody of a subject binding molecule can be used to target any low level expression target. Specific examples of low level expression targets that may be targeted by an IgM, IgA, IgG/IgM or IgG/IgA antibody of the subject binding molecules include, without limitation, EGFR, HER2, HER3, EpCAM, CEACAM, Gp100, MAGE1 and PD-L1. The GenBank Accession Numbers corresponding to the human protein sequences of these targets are provided in Table 4, below.

TABLE 4 Sequence information for low level expression targets SEQ ID Target Name NO. EGFR 136 HER2 137 HER3 138 EpCAM 139 CEACAM 140 Gp100 141 MAGE1 142 PD-L1 143

Low Affinity Targets

Aspects of the invention include binding molecules having an IgM, IgA, IgG/IgM or IgG/IgA antibody that binds to a low affinity target. Due to their higher avidity, the subject binding molecules are more potent than IgG antibodies. As such, the subject binding molecules can be employed in settings where a particular binding target has a low binding affinity, and where higher avidity is beneficial in facilitating binding between an antibody and a target. An antibody of a subject binding molecule can be used to target any low affinity target. Specific examples of low affinity targets that may be targeted by an IgM, IgA, IgG/IgM or IgG/IgA antibody of the subject binding molecules include, without limitation, NY-ESO-1, Sialyl Lewis X antigen, and Tn antigen. The GenBank Accession Numbers corresponding to the human protein sequences of NY-ESO-1 and Sialyl Lewis X antigen are provided in Table 5, below. The structure of Tn antigen is provided in FIG. 4.

TABLE 5 Sequence information for low affinity targets SEQ ID Target Name NO. NY-ESO-1 144 Sialyl Lewis X antigen 145

Hematologic Cancer Targets

Aspects of the invention include binding molecules having an IgM, IgA, IgG/IgM or IgG/IgA antibody that binds to a hematologic cancer target. Due to their higher avidity, the subject binding molecules are more potent than IgG antibodies. As such, the subject binding molecules can be employed in settings where a particular binding target is expressed at a low level, as is the case in certain hematologic cancers. The higher avidity of the subject binding molecules facilitates binding between an antibody and a target. An antibody of a subject binding molecule can be used to target any binding target, such as a low level expression target on a hematologic cancer cell. Specific examples of hematologic cancer targets that can be targeted by an IgM, IgA, IgG/IgM or IgG/IgA antibody of the subject binding molecules include, without limitation, CD19, CD20, CD22, CD33, CD38, CD52 and CD70. The GenBank Accession Numbers corresponding to the human protein sequences of these targets are provided in Table 6, below.

TABLE 6 Sequence information for hematologic cancer targets SEQ ID Target Name NO. CD19 146 CD20 147 CD22 148 CD33 149 CD38 150 CD52 151 CD70 152

Applications of Binding Molecules with Modified J-Chain

Binding molecules comprising a modified J-chain of the present invention have widespread therapeutic and diagnostic applications, including but not limited to the treatment of various cancers and immune diseases by modulating, among other things, the activity of a T-cell immune response. The subject binding molecules comprising a modified J-chain may broadly be used for the treatment of any of a variety of cancers. It is anticipated that any type of tumor and any type of tumor-associated antigen may be targeted by the subject binding molecules. Examples of cancer types include, without limitation, acute lymphoblastic leukemia, acute myelogenous leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal, gastric, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, and urinary bladder cancer. However, the skilled artisan will realize that tumor-associated antigens are known in the art for virtually any type of cancer.

In some embodiments, a J-chain of a subject binding molecule includes a binding moiety that antagonizes a T-cell inhibitory signaling pathway, and the antibody also antagonizes a T-cell inhibitory signaling pathway. Without being held to theory, the purpose of such a binding molecule is to block or decrease T-cell inhibitory signaling via both the antibody and the binding moiety on the J-chain. Such binding molecules provide a blockade or decrease of T-cell inhibitory signaling, thereby maintaining or increasing a T-cell immune response at a specific location, such as, e.g., the surface of a cancer cell. Due to their increased avidity, the subject IgM, IgA, IgG/IgM and IgG/IgA antibodies act more effectively as antagonists when directed to certain binding targets, such as members of a T-cell inhibitory signaling pathway, as described above. Such binding molecules find utility, for example, in the treatment of diseases wherein maintenance or activation of a T-cell immune response is desirable, such as, e.g., certain cancers and immune disorders. Such cancers include, but are not limited to, epithelial cancers as well as hematologic cancers.

Epithelial cancers that are suitable for treatment with the subject binding molecules having an antagonist antibody and an antagonist binding moiety on the J-chain include, without limitation, melanoma, non-small-cell lung, nasopharyngeal, colorectal, liver, urinary bladder, ovarian, gastric, esophageal, pancreatic, renal, thyroid or breast cancer, hormone receptor negative breast cancer, or triple negative breast cancer. Hematologic cancers that are suitable for treatment with the subject binding molecules having an antagonist antibody and an antagonist binding moiety on the J-chain include, without limitation, leukemia, lymphoma, myeloma, myelodysplastic syndrome, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma. In some embodiments, the subject binding molecules find use in the treatment of any of these conditions.

In some embodiments, a J-chain of a subject binding molecule includes a binding moiety that antagonizes a T-cell inhibitory signaling pathway, while the antibody agonizes a T-cell stimulatory signaling pathway. Without being held to theory, the purpose of such a binding molecule is to block or decrease T-cell inhibitory signaling via the J-chain moiety, while simultaneously maintaining or increasing T-cell stimulatory signaling via the antibody. Such binding molecules localize a blockade or decrease of T-cell inhibitory signaling (facilitated by the binding moiety on the J-chain) to the same site as the maintenance or activation of T-cell stimulatory signaling (facilitated by the antibody), thereby maintaining or increasing a T-cell immune response at a specific location, such as, e.g., the surface of a cancer cell. Due to their increased avidity, the subject IgM, IgA, IgG/IgM and IgG/IgA antibodies act more effectively as agonists when directed to certain binding targets, such as members of a T-cell stimulatory signaling pathway, as described above. Such binding molecules find utility, for example, in the treatment of diseases wherein maintenance or activation of a T-cell immune response is desirable, such as, e.g., certain cancers and immune disorders. Such cancers include, but are not limited to, epithelial cancers as well as hematologic cancers.

Epithelial cancers that are suitable for treatment with the subject binding molecules having an agonist antibody and an antagonist binding moiety on the J-chain include, without limitation, melanoma, non-small-cell lung, nasopharyngeal, colorectal, liver, urinary bladder, ovarian, gastric, esophageal, pancreatic, renal, thyroid or breast cancer, hormone receptor negative breast cancer, or triple negative breast cancer. Hematologic cancers that are suitable for treatment with the subject binding molecules having an agonist antibody and an antagonist binding moiety on the J-chain include, without limitation, leukemia, lymphoma, myeloma, myelodysplastic syndrome, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma. In some embodiments, the subject binding molecules find use in the treatment of any of these conditions.

In some embodiments, a J-chain of a subject binding molecule includes a binding moiety that antagonizes a T-cell inhibitory signaling pathway, while the antibody binds to a low level expression target. Without being held to theory, the purpose of such a binding molecule is to block or decrease T-cell inhibitory signaling via the binding moiety on the J-chain, while simultaneously binding to a low level expression target using the higher avidity of the IgM, IgA, IgG/IgM or IgG/IgA antibody. Such binding molecules provide localization of the blockade or decrease of T-cell inhibitory signaling at the site of a low level expression target, and find utility in the treatment of diseases wherein maintenance or activation of a T-cell immune response is desirable at the location of a low level expression target, such as, for example, certain cancers and immune disorders. For example, certain epithelial cancers are known to express tumor antigens that have a low level of expression, as described above. Such epithelial cancers include, without limitation, melanoma, non-small-cell lung, nasopharyngeal, colorectal, liver, urinary bladder, ovarian, gastric, esophageal, pancreatic, renal, thyroid or breast cancer, hormone receptor negative breast cancer, or triple negative breast cancer. In some embodiments, the subject binding molecules find use in the treatment of any of these conditions.

In some embodiments, a J-chain of a subject binding molecule includes a binding moiety that antagonizes a T-cell inhibitory signaling pathway, while the antibody binds to a low affinity target. Without being held to theory, the purpose of such a binding molecule is to block or decrease T-cell inhibitory signaling via the binding moiety on the J-chain, while simultaneously binding to a low affinity target using the higher avidity of the IgM, IgA, IgG/IgM or IgG/IgA antibody. Such binding molecules provide localization of the blockade or decrease of T-cell inhibitory signaling at the site of a low affinity target. As reviewed above, due to their increased avidity, the subject IgM, IgA, IgG/IgM and IgG/IgA antibodies, comprising a modified J-chain, are especially advantageous in situations where IgG antibodies bind to their target with low affinity. Thus, in some embodiments, the IgM, IgA, IgG/IgM and IgG/IgA antibodies described herein can comprise the binding domain of a therapeutic IgG antibody. Such binding molecules find utility in the treatment of diseases wherein maintenance or activation of a T-cell immune response is desirable at the location of a low affinity target, such as, for example, certain cancers and immune disorders. For example, certain epithelial cancers are known to express tumor antigens that have a low binding affinity, as described above. Such epithelial cancers include, without limitation, melanoma, non-small-cell lung, nasopharyngeal, colorectal, liver, urinary bladder, ovarian, gastric, esophageal, pancreatic, renal, thyroid or breast cancer, hormone receptor negative breast cancer, or triple negative breast cancer. In some embodiments, the subject binding molecules find use in the treatment of any of these conditions.

In some embodiments, a J-chain of a subject binding molecule includes a binding moiety that antagonizes a T-cell inhibitory signaling pathway, while the antibody binds to a target on a hematologic cancer cell. Without being held to theory, the purpose of such a binding molecule is to block or decrease T-cell inhibitory signaling via the binding moiety on the J-chain, while simultaneously binding to a hematologic cancer target using the higher avidity of the IgM, IgA, IgG/IgM or IgG/IgA antibody. Such binding molecules provide localization of the blockade or decrease of T-cell inhibitory signaling at the site of a hematologic cancer target, such as, e.g., on the surface of a hematologic cancer cell. Such binding molecules find utility in the treatment of hematologic cancers. For example, certain hematologic cancers are known to express tumor antigens at a low level, as described above. Such hematologic cancers include, without limitation, leukemia, lymphoma, myeloma, myelodysplastic syndrome, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma. In some embodiments, the subject binding molecules find use in the treatment of any of these conditions.

Examples of IgM, IgA, IgG/IgM, or IgG/IgA antibodies including a modified J-chain that antagonizes a T-cell inhibitory signaling pathway may include the binding regions of known IgG antibodies to tumor-associated antigens, such as, for example, blinatumomab (also known as MT103) (anti-CD19), CD19hA19 (anti-CD19, U.S. Pat. No. 7,109,304), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No. 7,251,164), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1 (anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No. 7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat. No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,541,440), hRS7 (anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat. No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496), the disclosures of which are expressly incorporated by reference herein.

Other antibodies that can provide binding regions for use in combination with a modified J-chain that antagonizes a T-cell inhibitory signaling pathway include, for example, abciximab (anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab (anti-EGFR), tositumomab (anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patent application Ser. No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab (anti-CD11a), GA101 (anti-CD20; Glycart Roche), atalizumab (anti-.alpha.4 integrin), omalizumab (anti-IgE); anti-TNF-.alpha. antibodies such as CDP571 (Ofei et al., 2011, Diabetes 45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (Thermo Scientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.), certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels, Belgium), adalimumab (Abbott, Abbott Park, Ill.), BENLYSTA.® (Human Genome Sciences); antibodies for therapy of Alzheimer's disease such as Alz 50 (Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab, solanezumab and infliximab; anti-fibrin antibodies like 59D8, T2G1s, MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202 (Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson & Johnson); trastuzumab (anti-HER2); tremelimumab (anti-CTLA4); urelumab (anti-CD137 (4-1BB)); vorsetuzumab (anti-CD70); duligotumab (anti-HER3); dacetuzumab (anti-CD40); varlilumab (anti-CD27); atezolizumab (anti-PD-L1); anti-MAGE1 antibodies such as MA454 (Thermo Scientific, Rockford, Ill.); anti-OX-40 antibodies such as ACT35 (Affymetrix eBioscience, San Diego, Calif.); anti-GITR antibodies such as 621 (BioLegend, San Diego, Calif.); anti-HVEM antibodies such as 122 (BioLegend, San Diego, Calif.); anti-TIM3 antibodies such as F38-2E2 (BioLegend, San Diego, Calif.); anti-LAG3 antibodies such as 3DS223H (Affymetrix eBioscience, San Diego, Calif.); anti-BTLA antibodies such as MIH26 (BioLegend, San Diego, Calif.); anti-VISTA antibodies such as MAB71261 (R&D Systems, Minneapolis, Minn.); anti-TIGIT antibodies such as MBSA43 (Affymetrix eBioscience, San Diego, Calif.); anti-CEACAM antibodies such as D14HD11 (abcam, Cambridge, Mass.); anti-Gp100 antibodies such as ab52058 (abcam, Cambridge, Mass.); anti-NY-ESO-1 antibodies such as E978 (Thermo Scientific, Rockford, Ill.); anti-Sialyl Lewis X antigen antibodies such as MAB2096 (EMD Millipore, Billerica, Mass.); anti-Tn antigen antibodies such as MA1-90544 (Thermo Scientific, Rockford, Ill.); anti-HIV antibodies such as P4/D10 (U.S. Pat. No. 8,333,971), Ab 75, Ab 76, Ab 77 (Paulik et al., 1999, Biochem Pharmacol 58:1781-90), as well as the anti-HIV antibodies described in U.S. Pat. Nos. 5,831,034, 5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob. Agents Chemother. 2006; 50(5):1773-9.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 and antagonizes a PD-L1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 and antagonizes a PD-L1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 and antagonizes a PD-L1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 and antagonizes a PD-L1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 and antagonizes a PD-L1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 and antagonizes a PD-L1-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD137 and agonizes a CD137-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD137 and agonizes a CD137-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD137 and agonizes a CD137-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD137 and agonizes a CD137-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LGA3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD137 and agonizes a CD137-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD137 and agonizes a CD137-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD137 and agonizes a CD137-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to OX40 and agonizes an OX40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to OX40 and agonizes an OX40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to OX40 and agonizes an OX40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to OX40 and agonizes an OX40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LGA3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to OX40 and agonizes an OX40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to OX40 and agonizes an OX40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to OX40 and agonizes an OX40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD40 and agonizes a CD40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD40 and agonizes a CD40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD40 and agonizes a CD40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD40 and agonizes a CD40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LGA3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD40 and agonizes a CD40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD40 and agonizes a CD40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD40 and agonizes a CD40-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GITR and agonizes a GITR-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GITR and agonizes a GITR-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GITR and agonizes a GITR-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GITR and agonizes a GITR-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LGA3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GITR and agonizes a GITR-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GITR and agonizes a GITR-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GITR and agonizes a GITR-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD27 and agonizes a CD27-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD27 and agonizes a CD27-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD27 and agonizes a CD27-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD27 and agonizes a CD27-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LGA3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD27 and agonizes a CD27-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD27 and agonizes a CD27-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD27 and agonizes a CD27-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HVEM and agonizes an HVEM-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HVEM and agonizes an HVEM-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HVEM and agonizes an HVEM-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HVEM and agonizes an HVEM-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LGA3-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HVEM and agonizes an HVEM-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HVEM and agonizes an HVEM-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In a specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HVEM and agonizes an HVEM-mediated T-cell stimulatory signaling pathway has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers and hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EGFR has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EGFR has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EGFR has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EGFR has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EGFR has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EGFR has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EGFR has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER2 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER2 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER2 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER2 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER2 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER2 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER2 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER3 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER3 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER3 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER3 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER3 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER3 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to HER3 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EPCAM has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EPCAM has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EPCAM has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EPCAM has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EPCAM has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EPCAM has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to EPCAM has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CEACAM has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CEACAM has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CEACAM has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CEACAM has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CEACAM has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CEACAM has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CEACAM has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GP100 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GP100 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GP100 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GP100 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GP100 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GP100 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to GP100 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to MAGE1 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to MAGE1 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to MAGE1 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to MAGE1 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to MAGE1 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to MAGE1 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to MAGE1 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to PD-L1 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to NY-ESO-1 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to NY-ESO-1 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to NY-ESO-1 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to NY-ESO-1 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to NY-ESO-1 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to NY-ESO-1 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to NY-ESO-1 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Sialyl Lewis X antigen has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Sialyl Lewis X antigen has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Sialyl Lewis X antigen has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Sialyl Lewis X antigen has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Sialyl Lewis X antigen has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Sialyl Lewis X antigen has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Sialyl Lewis X antigen has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Tn antigen has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Tn antigen has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Tn antigen has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Tn antigen has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Tn antigen has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Tn antigen has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to Tn antigen has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to epithelial cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD19 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD19 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD19 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD19 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD19 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD19 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD19 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD20 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD20 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD20 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD20 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD20 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD20 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD20 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD22 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD22 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD22 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD22 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD22 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD22 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD22 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD33 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD33 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD33 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD33 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD33 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD33 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD33 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD38 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD38 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD38 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD38 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD38 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD38 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD38 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD52 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD52 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD52 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD52 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD52 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD52 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD52 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to hematologic cancers.

In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD70 has a binding moiety on the J-chain that binds to CTLA4 and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD70 has a binding moiety on the J-chain that binds to PD-1 and antagonizes a PD-1-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD70 has a binding moiety on the J-chain that binds to TIM3 and antagonizes a TIM3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD70 has a binding moiety on the J-chain that binds to LAG3 and antagonizes a LAG3-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD70 has a binding moiety on the J-chain that binds to BTLA and antagonizes a BTLA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD70 has a binding moiety on the J-chain that binds to VISTA and antagonizes a VISTA-mediated T-cell inhibitory signaling pathway. In one specific embodiment, a binding molecule whose IgM, IgA, IgG/IgM, or IgG/IgA antibody binds to CD70 has a binding moiety on the J-chain that binds to TIGIT and antagonizes a TIGIT-mediated T-cell inhibitory signaling pathway. Such binding molecules find use in the treatment of cancers, including but not limited to hematologic cancers.

It is to be understood that an IgM, IgA, IgG/IgM or IgG/IgA antibody that binds to any of the listed tumor antigens can be combined with a modified J-chain with any of the binding specificities listed herein to create a binding molecule. Thus, any antibody target listed herein can be combined with any modified J-chain target listed herein.

While certain preferred embodiments are specifically referred to herein, it is to be understood that IgM, IgA, IgG/IgM and IgG/IgA antibodies with binding specificity to any target, such as any tumor antigen, comprising a modified J-chain with a binding moiety that binds to any target that antagonizes a T-cell inhibitory signaling pathway are contemplated and are within the scope of the present invention. FIG. 7 provide a list of antibody targets and targets for the binding moiety of a J-chain. Any of the antibody targets listed in the left column of FIG. 7 can be combined with any of the binding moiety targets for the J-chain listed in the right column of FIG. 7.

In a preferred embodiment, an IgM, IgA, IgG/IgM or IgG/IgA antibody binds to one or more of the tumor targets listed herein, while the J-chain comprises a binding moiety that antagonizes a T-cell inhibitory signaling pathway.

In one preferred embodiment, a J-chain of a subject binding molecule includes a binding moiety that is an scFv, and that antagonizes a T-cell inhibitory signaling pathway by binding to a target in the pathway.

In one preferred embodiment, the binding moiety on the J-chain is an scFv that binds to CTLA4 (i.e., is an anti-CTLA4scFv) and antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway.

In one preferred embodiment, a binding molecule includes an IgM antibody that binds to PD-L1, and the binding moiety on the J-chain is an anti-CTLA4scFv that antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway.

In one preferred embodiment, a binding molecule includes an IgM antibody that binds to PD-1, and the binding moiety on the J-chain is an anti-CTLA4scFv that antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway.

In one preferred embodiment, a binding molecule includes an IgM antibody that binds to TIM3, and the binding moiety on the J-chain is an anti-CTLA4scFv that antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway.

In one preferred embodiment, a binding molecule includes an IgM antibody that binds to LAG3, and the binding moiety on the J-chain is an anti-CTLA4scFv that antagonizes a CTLA4-mediated T-cell inhibitory signaling pathway.

In all embodiments, a binding moiety on a modified J-chain may be introduced before or after the J-chain. Thus, for example, a modified J-chain with an scFv binding moiety that antagonizes a T-cell inhibitory signaling pathway by binding to CTLA4 may have an anti-CTLA4scFv-J or a J-anti-CTLA4scFv configuration. A schematic illustration of both of these configurations is shown in FIG. 5.

Due to their increased avidity, the subject binding molecules are superior relative to bispecific IgG antibodies. For example, as a result, they are suitable for targeting low level expression targets, such as Rituxan-resistant Burkitt lymphoma cells characterized by a low level of CD20 expression. In addition, the IgM, IgA, IgG/IgM and IgG/IgA antibodies herein comprising a modified J-chain have greatly enhanced potency relative to bispecific IgG antibodies.

Pharmaceutical Compositions of Antibodies with Modified J-Chain

For therapeutic uses, a subject binding molecule can be formulated into pharmaceutical compositions. A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the target disease or condition and the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

The compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and/or dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier preferably is an isotonic buffered saline solution.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Further details of the invention are illustrated by the following, non-limiting Examples.

Example 1: Preparation of a Bispecific Anti-PD-L1 Antibody Comprising a Modified J-Chain with a Binding Moiety that Binds to CTLA4

1. Generation of DNA constructs with designed mutations:

    • a. DNA construct synthesis. All the DNA constructs with designed mutations are synthesized by commercial vendors (Genescript), with compatible restriction sites at both ends for subcloning into respective expression vectors.
    • b. Constructing expression vectors. The synthesized DNA constructs are re-suspended in Tris-EDTA buffer at 1 μg/ml. DNA (1 μg) is subjected to enzyme digestion and the synthesized gene is separated from the carrier plasmid DNA by electrophoresis. The digested DNA is ligated to pre-digested plasmid DNA (pCAGGS for J-chain, Gene 108 (1991) 193-200) by standard molecular biology techniques. The ligated DNA is transformed into competent bacteria and plated on LB plates with multiple selective antibiotics, Several bacterial colonies are picked and DNA preparations are made by standard molecular biology techniques. The prepared DNA are verified by sequencing. Only the bacterial clones with 100% match of DNA sequence with the designed DNA sequence are used for plasmid DNA preparation and subsequently for cell transfection.
      • i. The first construct is composed of a scFv version of anti-CTLA4 fused with N-terminus of human J-chain (CTLA4 scFv-15 aa Linker-J). The amino acid sequence of this construct (Y15J) is:

(SEQ ID NO: 2) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTF ISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTG WLGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGE RATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKGGGGSGGGG SGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPL NNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDE DSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD
      • ii. The second construct is composed of a scFv of anti-CTLA4 fused with C-terminus of human J-chain (J-15 aa Linker-CTLA4 scFv). The amino acid sequence of this construct (J15Y) is:

(SEQ ID NO: 3) QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENI SDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATET CYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGGSGGGGSGGG GSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWV TFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAR TGWLGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSP GERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSG SGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

Both of these constructs are designed to enable integration of the J-chain into an IgM that is specific for PD-L1.

IgM heavy chain: This heavy chain construct has a full length μ chain with a Vh region derived from an anti-PD-L1 antibody:

(SEQ ID NO: 4) MDPKGSLSWRILLFLSLAFELSYGEVQLVESGGGLVQPGGSLRLSCAASG HTSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKN TAYIQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAGSASAPTLF PLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSV LRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAEL PPKVSVFVPPRDGFFGNPRKSKLICQATGESPRQIQVSWLREGKQVGSGV TTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQN ASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISW TRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTD LPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGE TYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

This heavy chain construct has a molecular weight about 64 kD and when co-expressed with light chain, the resultant IgM is designed to bind to PD-L1 over-expressed on tumor cells.

    • d. Light chain for this bispecific IgM is derived from an anti-PD-L1 antibody:

(SEQ ID NO: 5) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQ GTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

The light chain construct has a molecular weight about 24 kD and when co-expressed with the appropriate heavy chain (SEQ ID NO: 4) is designed to bind to PD-L1 on tumor cells.

2. Protein expression, purification and characterization

    • a. Transfection. Heavy, Light and Modified J-chain DNA is transfected into CHO cells. DNA for expression vectors are mixed typically in 1:1:1 ratio with PEI and then added to CHO-S cells. PEI transfection with CHO-S cells is conducted according to established techniques (see “Biotechnology and Bioengineering, Vol 87, 553-545”).
    • b. Immunoprecipitation
      • i. CAPTURESELECT® IgM (BAC, Thermo Fisher). IgM proteins from transfected CHO cell supernatants are partially purified by immunoprecipitation with CAPTURESELECT® IgM affinity matrix according to manufacturers' protocol (GE Life Sciences). After incubation at room temperature for 2 hours, the affinity matrix is separated from the supernatant by centrifugation. The matrix is further washed with PBS for 3 times before the PBS is carefully removed. The captured protein is eluted from the matrix by incubating with NuPage LDS protein buffer (Life Technology) for 5 minutes.
    • c. Gel electrophoresis
      • i. Non-reducing SDS PAGE separates native IgM and its mutant forms according to size. Pentameric IgM, composed of homodimeric heavy and light chains, produces a protein band of approximately 1,000.000 molecular weight. NuPage LDS Sample Buffer (Life Technologies) is added to IgM protein samples at 25 C for 30 minutes before loading onto the gel. NativePage Novex 3-12% Bis-Tris Gel (Life Technologies) is used with Novex Tris-Acetate SDS Running Buffer (Life Technologies) Run gel until the dye front reaches the bottom of the gel. (FIG. 8)
      • ii. Reducing SDS-PAGE. NuPage LDS sample buffer (Life Technologies) and NuPage reducing agent dithiothreitol (Life Technologies) are added to IgM protein samples and heated to 80° C. for 10 minutes before loading on NuPage Novex 4-12% Bis-Tris Gel (Life Technologies). NuPage MES SDS Running Buffer (Life Technologies) is used for gel electrophoresis. Gels are run until the dye front reaches the bottom of the gel. After electrophoresis is complete, remove gel from apparatus and stain the gel using Colloidal Blue Staining (Life Technologies).
      • iii. Western Blot Detection. After electrophoresis is complete, remove gel from XCELL SURELOCK® Mini-Cell. Transfer to PVDF membrane at 30 volts for 1 hour (refer to Life Technologies' manual). Block with 20 ml 3% BSA in PBST at 25 C for 1 hour.

For anti-J-chain Western blot, add anti-J (SP105, Thermo Fisher) at 1:500 in 3% BSA in PBST overnight at 4 C. Wash with PBST four times at room temperature. Add HRP-Goat anti rabbit IgG (Jackson Immunology) at 1:5,000 in 3% BSA in PBST for 1 hour at room temperature. Wash with PBST 4 times at room temperature. Add 10 ml of HRP chemiluminescent substrate (Thermo Fisher) for 10 minutes before exposing the blot to film. Anti-J-chain antibody only reacts with IgM which is co-expressed with either unmodified J-chain or modified J-chain. As shown in FIG. 8, the anti-PD-L1 IgM with either the wild type J-chain or the modified J-chain carrying the anti-CTLA4 scFv (Y15J) is clearly assembled correctly.

Example 2: Anti-CD20 IgMs Carrying Anti-CD3 scFv Binding Moiety on their J-Chains can Activate T-Cells Only in the Presence of CD20 Positive B-Cells

This example illustrates the preparation and characterization of an IgM molecule comprising a modified J-chain. Specifically, this example describes the preparation of the molecular cloning, expression and purification of an IgM antibody targeting a B-cell antigen (CD20) and a modified J-chain that comprises a binding moiety that binds to CD3, to demonstrate production of a bispecific IgM and measurement of the functional activity in a relevant system. The DNA corresponding to the heavy, light and J-chain sequences below was prepared using the methods as described in Example 1.

Amino acid sequence of IgM Light chain sequence of an anti-CD20 antibody:

(SEQ ID NO: 6) MDMRVPAQLLGLLLLWLRGARCQIVLSQSPAILSASPGEKVTMTCRASSS VSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVE AEDAATVYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVIKSFNRGEC

Amino acid sequence of IgM Heavy chain sequence of an anti-CD20 antibody:

(SEQ ID NO: 7) MGWSYIILFLVATATGVHSQVQLQQPGAELVKPGASVKMSCKASGYTFTS YNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM QLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSGSASAPTLFP LVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVL RGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELP PKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVT TDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNA SSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCVTDLTTYDSVTISWTR QNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLP SPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADV FVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETY TCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

Amino acid sequence of J chain sequence for V15J:

(SEQ ID NO: 8) MGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFIS YTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYM ELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSG GGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRL IYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPT FGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRS SEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPT EVELDNQTVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMV ETALTPDACYPD

Amino acid sequence of J-chain sequence for J15V:

(SEQ ID NO: 9) MKNHLLFWGVLAVFIKAVHVKAQEDERIVINDNKCKCARITSRIIRSSED PNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVE LDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETA LTPDACYPDGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASG YTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSA STAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGK APKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWS SNPPTFGGGTKLEIK

Amino acid sequence of J-chain sequence for O15J:

(SEQ ID NO: 10) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGY INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPG EKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGS GTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIKGGGGSGGGGS GGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLN NRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDED SATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGSEQKL ISEEDLNSAVDHHHHHH

Amino acid sequence of J-chain sequence for J15O:

(SEQ ID NO: 11) QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENI SDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATET CYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGGSGGGGSGGG GSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWI GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYSLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSAS PGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGS GSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIKEQKLISEE DLNSAVDHHHHHH

The DNA corresponding to these heavy and light chains as well as that corresponding to either the wild-type (wt) J-chain (FIG. 3), V15J or J15V J-chain sequences shown above were co-transfected into HEK293 cells and proteins expressed and purified using the camelid resin as described before. As shown in FIG. 9, Panel A, all four proteins express well. The anti-CD20 IgM hexamer without J-chain is clearly resolved from the J-chain containing pentamers for the IgM pentamer with the wild type J-chain as well as for the bispecific IgM's where the anti-CD3 scFv is linked to the J-chain in either orientation (FIG. 9, Panel A).

Purified proteins were analyzed for T-cell activation using a commercially available Luciferase reporter gene based kit (Promega). Briefly, purified protein was added to 7500 Ramos and 25000 engineered Jurkat cells (Promega CS176403) in 40 uL RPMI with 10% FBS. Mixture was incubated for 5 h 37 C with 5% CO2. Cells were mixed with lysis buffer containing luciferin to measure luciferase reporter activity. Light output was measured by EnVision plate reader and analyzed by Prism software. As shown in FIG. 9, Panel B, only the antibodies that carried the CD3 specific scFv binding moiety on the J-chain are able to show dose dependent activation, whereas the IgM antibody lacking the modified J-chain or the IgG are unable to show any signal in this assay.

Example 3: IgM Binds Better to Low Abundance Targets than IgG

Roughly 30×10{circumflex over ( )}3 cells per well were loaded in FACS buffer (2% FBS/PBS) in a V-bottom plate. The plates were spun and supernatant aspirated. Serially diluted antibodies in FACS buffer were added in a volume of 50 μL to the cells and incubated for 30 minutes on ice. The cells were then washed with 150 μL of FACS buffer and then pelleted at 1200 rpm for 5 minutes at room temperature. The supernatant was aspirated and 50 μL of relevant secondary at 1 μg/mL was added to each well. The plate was further incubated for 30 minutes on ice. The cells were then washed with 150 μL of FACS buffer, pelleted at 1200 rpm for 5 minutes at room temperature, supernatant aspirated and 60 μL of 7-AAD FACS buffer (1:100) added. After a brief incubation (5 min), data were acquired, gating for AAD negative cells, on a FACS calibur. Binding data was analyzed using GraphPad Prism software. As shown in FIG. 10, the anti-PD-L1 IgM and IgG bind comparably to the high PD-L1 expressing cells (Promega transfected CHO cell line). On a low PD-L1 expressing cell line (Arent), the anti-PD-L1 IgM is seen to bind significantly better than the anti-PD-L1 IgG (more than 10× better on a molar basis).

Example 4: Anti-PD-L1 IgM has Functional Effect on T-Cell Activation Better than IgG

Anti-PD-L1 IgG and IgM antibodies were characterized for B cell dependent activation of T cells using a reporter cell line (Promega). T cell activation increases the NFAT dependent luciferase expression, engineered into Jurkat cells. The enhanced expression can be measured after lysis using a luminescence readout.

Briefly, purified protein was added to 7500 Ramos and 25000 engineered Jurkat cells (Promega CS176403) in 40 μL RPMI with 10% FBS. Mixture was incubated for 5 h 37 C with 5% CO2. Cells were mixed with lysis buffer containing luciferin to measure luciferase reporter activity. Light output was measured by EnVision plate reader and analyzed by Prism software.

As shown in FIG. 11, the anti-PD-L1 IgM with or without a J-chain can inhibit PD-L1:PD-1, interaction leading to activation of the reporter cell and increased luminescence. It is also clear that on a molar basis the anti-PD-L1 IgM is able to activate T-cells better than the corresponding IgG.

Example 5: Bispecific Anti-PD-L1 IgM with Anti-CD3 scFv Fused to J-Chain can can Activate T-Cells in the Presence of PD-L1 Expressing Cells

This example illustrates the preparation and characterization of an IgM molecule comprising a modified J-chain. Specifically, this example describes the preparation of the molecular cloning, expression and purification of an IgM antibody targeting a PD-L1 and a modified J-chain that comprises a binding moiety that binds to CD3, to demonstrate production of a bispecific IgM and measurement of the functional activity in a relevant system. The DNA corresponding to the heavy, light and J-chain sequences below was prepared using the methods as described in Example 1.

Amino acid sequence of IgM Light chain sequence of an anti-PD-L1 antibody:

(SEQ ID NO: 106) MDMRVPAQLLGLLLLWLRGARCQIVLSQSPAILSASPGEKVTMTCRASSS VSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVE AEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Amino acid sequence of IgM Heavy chain sequence of an anti-PD-L1 antibody:

(SEQ ID NO: 104) MDPKGSLSWRILLFLSLAFELSYGEVQLVESGGGLVQPGGSLRLSCAASG FTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSK NTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAGSASAPTL FPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAE LPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSG VTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQ NASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTIS WTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHT DLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSP ADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTG ETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

Amino acid sequence of J chain sequence for V15J:

(SEQ ID NO: 108) MGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFIS YTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYM ELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSG GGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRL IYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPT FGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRS SEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPT EVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMV ETALTPDACYPD

The DNA corresponding to these heavy and light chains as well as that corresponding to either the wild-type (wt) J-chain (FIG. 12), V15J sequences shown above were co-transfected into HEK293 cells and proteins expressed and purified using the camelid resin as described before. As shown in FIG. 9, Panel A, all four proteins express well. The anti-PD-L1 hexamer without J-chain is clearly resolved from the J-chain containing pentamers for the IgM pentamer with the wild type J-chain as well as for the bispecific IgM's where the anti-CD3 scFv is linked to the J-chain (FIG. 12, Panel A).

Anti-PD-L1 IgM antibodies with or without the J-chains described above were characterized for release of PD-L1 expressing tumor cell dependent inactivation of T cells using a reporter cell line (Promega). T cell activation increases the NFAT dependent luciferase expression, engineered into Jurkat cells. The enhanced expression can be measured after lysis using a luminescence readout.

Briefly, purified protein was added to 7500 Ramos and 25000 engineered Jurkat cells (Promega CS176403) in 40 μL RPMI with 10% FBS. Mixture was incubated for 5 h 37 C with 5% CO2. Cells were mixed with lysis buffer containing luciferin to measure luciferase reporter activity. Light output was measured by EnVision plate reader and analyzed by Prism software.

As shown in FIG. 12 Panel B, the anti-PD-L1 IgM with or without a J-chain can inhibit PD-L1:PD-1, interaction leading to activation of the reporter cell and increased luminescence. It is also clear that addition of the CD3 binding J-chain fusion does not interfere with the ability of this IgM to block PD-1:PD-L1 interaction.

Example 6: Bispecific Anti PD-L1 IgM with Anti-CD3 scFv Fused to J-Chain can Use T-Cells to Kill PD-L1 Expressing Cells

Engagement of effector T-cells by bispecific IgM antibodies with a modified J-chain is expected to greatly enhance killing of the target B-cell populations compared to the IgM carrying no J-chain or the wild type J-chain. To test cell killing in co-culture, we performed a cell killing assay. Antibody doses were incubated with Oregon Green 488 labeled PD-L1+ cells (either high expressing HDML2 or low expressing SUPHD1) and purified CD8+ effector cells. As shown in FIG. 14, the bispecific IgM carrying a CD3 binding scFv on its J-chain is able to cause complete killing of PD-L1 expressing cells. Complete killing of target cells by bispecific IgM is observed at concentrations as low as 2 pM.

Example 7: Half-Life Extended Anti-PD-L1 IgM with Albumin or Albumin Binding scFv Fused to J-Chain can be Made and Still Block PD-1:PD-L1 Interaction

The half-life of IgMs in human plasma is estimated to be around 2-3 days and shorter still in mice. This is significantly shorter than for IgGs, which interact with the neonatal Fc receptor (FcRn) and are recycled after endocytosis enabling a much longer half-life of roughly 21 days. In order to increase the half-life of our anti-PD-L1 IgMs, we took advantage of the fact that we can tether scFvs to either terminus of the J-chain without significantly altering the effector functions of IgMs such as CDC.

There are several approaches that have been described in the art to enable half-life extension of biologics. These include tethering of mutants of human serum albumin (Andersen et al, JBC VOL. 289, NO. 19, pp. 13492-13502, 2014), peptides (Dennis et al, J. Biol. Chem. 2002, 277:35035-35043) or scFvs that can bind human serum albumin (Muller et al mAbs 4:6, 673-685; 2012),

Modified J-Chain Sequences are Provided Herein.

Expression and assembly of this ABD-J-chain fusion or HSA-J-chain fusion into IgMs was tested using the IgM sequence described in Example 1 (FIG. 15 Panel A). In addition, we verified that fusion of ABD or HSA to J-chain does not perturb the blockade activity on anti-PD-L1 IgM on target cell lines carrying PD-L1 on their surface as described in Example 5.

Example 8: Anti-PDL-1 IgM (S70) with Anti-CTLA-4 J-Chain (Y15J) can be Made and Retains Activity of Both Arms

Expression and assembly of this anti-CTLA-4 scFv-J-chain fusion (Y15J) into S70 IgMs was tested using the IgM sequence described in Example 1 (FIG. 16 Panel A). In addition, we verified that fusion of this scFv to J-chain does not perturb the blockade activity on anti-PD-L1 IgM on target cell lines carrying PD-L1 on their surface (FIG. 16 Panel B) as described in Example 5.

To verify that the CTLA-4 binding scFv on the J-chain retains binding to CTLA-4, bispecific anti PDL1 IgM with antiCTLA4 linked to amino terminus of J chain was expressed in Expi293 and affinity purified by CAPTURESELECT® IgM as described in previous examples. Purified Fc fusion protein of Human CTLA4 was immobilized to Fortebio sensors using amine reactive chemistry in sodium Acetate at pH 6.0. Anti CTLA4 IgG (BioLegend A3.B10.G1) binds to immobilized CTLA4 sensor with KD of 2 nM. Binding rates of anti CTLA4 scFv are similar to that of IgG. As expected, the dissociating rate of the monovalent anti CTLA4 scfv is faster than that of the IgG (FIG. 17 Panels A and B).

Example 9: Demonstration that an Anti-TNF Receptor Superfamily (DR5) Antibody can have Super-Agonist Activity with Dramatic Improvement Over IgG

The multivalent nature of IgA or IgM molecules presents a useful tool for application to specific biological systems in which multiple components necessarily must be bound simultaneously to transmit biological signals. For instance, many receptor proteins on the surface of eukaryotic cells require the simultaneous activation of multiple monomers or subunits to achieve activation and transmission of a biological signal across a cell membrane, to the cytoplasm of the cell.

One such system of cell surface protein receptors requiring multimerization prior to, or commensurate with, activation is found in the Tumor Necrosis Factor (TNF) superfamily of receptor proteins. Within this superfamily of receptor proteins are members which, upon activation, transmit a signal to the nucleus of the cell causing apoptosis. Other family members of this superfamily cause activation of NF-κB, apoptosis pathways, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), and c-Jun N-terminal kinase (JNK). Non-limiting examples of TNF superfamily receptor members that regulate apoptosis of a cell when activated include the following: TNFR1 (DR1), TNFR2, CD40 (p50), Fas (CD95, Apo1, DR2), CD30, 4-1BB (CD137, ILA), TRAILR1 (DR4, Apo2), DR5 (TRAILR2), TRAILR3 (DcR1), TRAILR4 (DcR2), OPG (OCIF), TWEAKR (FN14), LIGHTR (HVEM), DcR3, DR3, EDAR, and XEDAR. (See, Aggarwal et al., Blood, 119:651-665, 2012).

More particularly, it is postulated that activation of the TNF superfamily receptor protein members mentioned above requires that at least three non-interacting receptor monomers be cross-linked, e.g., by a ligand, to form a stabilized receptor trimer, resulting in signal transduction across the cell membrane. Clustering of these TNF superfamily receptor protein trimers into “rafts” of trimers has been observed and has been postulated to lead to more effective activation of this TNF superfamily receptor protein-dependent signaling cascade. (See, Valley et al., J. Biol. Chem., 287(25):21265-21278, 2012). Additional modes of activation have been discussed. (See, for instance, Lewis et al., Biophys. J., 106(6):L21-L24, 2014) (FIG. 12).

Amino acid sequence of IgM Heavy chain sequence of an anti-DR5 antibody:

(SEQ ID NO: 12) EVQLVQSGGGVERPGGSLRL SCAASGFTFD DYGMSWVRQA PGKGLEWVSG INWNGGSTGY ADSVKGRVTI SRDNAKNSLY LQMNSLRAED TAVYYCAKIL GAGRGWYFDL WGKGTTVTVS SASTKGPSVF PLAPSSKSTS GGFAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

Amino acid sequence of IgM Light chain sequence of the anti-DR5 antibody:

(SEQ ID NO: 13) SSELTQDPAV SVALGQTVRI TCQGDSLRSY YASWYQQKPG QAPVLVIYGK NNRPSGIPDR FSGSSSGNTA SLTITGAQAE DEADYYCNSR DSSGNHVVFG GGTKLTVLGQ PKAAPSVTLF PPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAG VETTTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS

Amino acid sequence of wild-type J-chain sequence:

(SEQ ID NO: 1) QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENI SDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATET CYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

The DNA corresponding to these heavy and light chains as well as that corresponding to the wild-type (wt) J-chain sequence were co-transfected into HEK293 cells, and proteins were expressed and purified using the camelid resin as described above. To test cytotoxicity of the purified proteins, a cell viability assay was carried out using COLO205 cells. Briefly, 5000 cells per well were seeded into a 96 well white plate in 25 μL. Antibodies were diluted in phenol-red free medium in a volume of 25 μL and the dilution added to the plate containing the cells. After incubating at 37 C for 24 hours, cell viability was measured using Cell-Titer Glo reagent (Promega). As shown in FIG. 13, the anti-DR5 IgM and IgM+wt J-chain antibodies show dramatically improved cytotoxic effect (greater than 1,000 fold) compared to the corresponding IgG. This underscores the ability of IgM pentamers/hexamers to carry out super-agonist activity on TNF receptor superfamily targets.

Example 10: Anti-CDIM Antibody (IGM-55.5)

IGM-55.5 is a recombinant monoclonal human IgM antibody derived from a natural monoclonal antibody 216 isolated at Stanford University from the splenocytes of a patient with Non-Hodgkin's lymphoma. HuMab 216 was previously used in a B-cell acute lymphoblastic leukemia phase I trial and was demonstrated to be well tolerated with significant decrease in peripheral blasts observed (Liedtke et al, Haematologica, 2012). IGM-55.5 has been re-engineered to be more specific to a carbohydrate determinant as an epitope on normal human B cells as well as B-cell lymphoma and B-progenitor lymphoblasts and therefore to be a potential therapeutic for advanced B cell malignancies, especially indicated for rituximab resistant or refractory patients. The amino acid sequences of the IGM-55.5 light chain and heavy chain are provided below.

IGM-55.5 heavy chain. This heavy chain construct has a full length μ chain for IGM-55.5 which binds CDIM on the surface of B-cells:

(SEQ ID NO: 14) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGE INHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRM AWGASVNFDYWGQGTLVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCL AQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVM QGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRK SKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVT STLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPP SFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPN ATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRP DVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVT SAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTV DKSTGKPTLYNVSLVMSDTAGTCY

This heavy chain construct has a molecular weight about 64 kD and when co-expressed with light chain, the resultant IgM is able to bind to CDIM positive B cells.

Light chain for IGM-55.5 known as IGM-55.5, which binds CDIM (cell death inducing molecule) on the surface of B-cells:

(SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQ GTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

The light chain construct has a molecular weight about 24 kD and when co-expressed with the appropriate heavy chain (SEQ ID NO: 14) is able to bind to CDIM positive B cells. IGM-55.5 can be made with a modified J-chain, as described herein, and any of the above-described binding moieties can be added to the J-chain.

TABLE 7 Sequence Summary SEQ ID NO: Short Name Sequence  27 Rituximab VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTT VTVSA  28 Rituximab SYNMH HCDR1  29 Rituximab AIYPGNGDTSYNQKFKG HCDR2  30 Rituximab STYYGGDWYFNV HCDR3  31 Rituximab VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYTHWFQQKPGSSPKPWIYATSNLASGVP VRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKR  32 Rituximab RASSSVSYIH LCDR1  33 Rituximab ATSNLAS LCDR2  34 Rituximab QQWTSNPPT LCDR3  35 900 VH EVQLVESGGG LVQPGGSLRL SCAASGYTFT SYNMHWVRQA PGKGLEWVGA IYPGNGDTSY NQKFKGRFTI SVDKSKNTLY LQMNSLRAED TAVYYCARVV YYSNSYWYFD VWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT  36 900HCDR3 VVYYSNSYWYFDV  37 900VL DIQMTQSPSS LSASVGDRVT ITCRASSSVS YMHWYQQKPG KAPKPLIYAP SNLASGVPSR FSGSGSGTDF TLTISSLQPE DFATYYCQQW SFNPPTFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL  38 900LCDR1 RASSSVSYMH  39 900LCDR2 APSNLAS  40 900LCDR3 QQWSFNPPT  41 125 VH EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDT SYNQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYVGGDWQFDVWGKGTT VTVSS  42 125HCDR2 AIYPLTGDTSYNQKSKL  43 125HCDR3 STYVGGDWQFDV  44 125 VL EIVLTQSPGTLSLSPGERATLSCRASSSVPYIHWYQQKPGQAPRLLIYATSALASGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSNPPTFGQGTKLEIK  45 125LCDR1 RASSSVPYIH  46 125LCDR2 ATSALAS  47 125LCDR3 QQWLSNPPT  48 844 VH #2 QVQLQQPGAELKKPGASVKVSCKASGYTFTSYNMHWVKQTPGRGLEWTGAIYPGNGDT SYNQKFKGKTTLTADKSSSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGAGTT VTVSA  49 844 VH #3 QVQLQQPGAELKKPGASVKVSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDT SYNQKFKGKTTLTADKSSSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGAGTT VTVSA  50 844 VL #5 QIVLSQSPAIITASPGEKVTMTCRASTSASYIHWFQQKPTSSPKPWIYATSNLASGVP SRFSGSGSGTTYSMTISSLEAEDAATYYCQQWTSNPPTFGGGTKLEIK  51 844 VL #5 RASTSASYIH LCDR1  52 844 VL #6 QIVLSQSPAIITASPGEKVTMTCRASTSVSYIHWFQQKPTSSPKPWIYATSNLASGVP SRFSGSGSGTTYSMTISSLEAEDAATYYCQQWTSNPPTFGGGTKLEIK  53 844 VL #6, #7 RASTSVSYIH LCDR1  54 844 VL #7 QIVLSQSPAIITASPGEKVTMTCRASTSVSYIHWFQQKPGSSPKPWIYATSNLASGVP SRFSGSGSGTTYSMTISSLEAEDAATYYCQQWTSNPPTFGGGTKLEIK  55 844 VL #8 QIVLSQSPAIITASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVP SRFSGSGSGTTYSMTISSLEAEDAATYYCQQWTSNPPTFGGGTKLEIK  56 844 VH #10 EVQLQQSGAELKKPGASVKVSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDT SYNQKFKGKTTLTADKSSSTAYMELSSLRSEDTAVYYCARSNYYGSSYWFFDVWGTGT TVTVSS  57 844 VH #10 SNYYGSSYWFFDV HCDR3  58 844 VL #12 DIVLTQSPAIITASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVP SRFSGSGSGTTYSMTISSLEAEDAATYYCQQWSFNPPTFGGGTKLEIK  59 844 VL #12 RASSSVNYMD LCDR1  60 844 VL #12 QQWSFNPPT LCDR3  61 164 VH QVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVKQAPGQGLEWIGAIYPGNGDT SYNQKFKGKATLTADESTNTAYMELSSLRSEDTAFYYCARSTYYGGDWYFDVWGQGTT VTVSS  62 164 VH STYYGGDWYFDV HCDR3  63 164 VL MGWSCIILFLVATATGVHSDIQLTQSPSSLSASVGDRVTMTCRASSSVSYIHWFQQKP GKAPKPWIYATSNLASGVPVRFSGSGSGTDYTFTISSLQPEDIATYYCQQWTSNPPTF GGGTKLEIK  64 1.5.3 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSITTAYLQWSSLKASDTAMYYCARHPSYGSGSPNFDYWGQGT LVTVSS  65 1.5.3 HCDR1 GYSFTSYWIG  66 1.5.3 HCDR2 IIYPGDSDTRYSPSFQG  67 1.5.3 HCDR3 HPSYGSGSPNFDY  68 1.5.3 VL DIVMTQTPLSSPVTLGQPASISCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKSN RFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQATQFPLTFGGGTKVEIK  69 1.5.3 LCDR1 RSSQSLVYSDGNTYLS  70 1.5.3 LCDR2 KISNRFS  71 1.5.3 LCDR3 VQATQFPLT  72 human IgM GCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCG constant TGGCCGTTGGCTGCCTCGCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAA region DNA ATACAAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGG GGCAAGCACGCAGCCACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCA CAGACGAACACGTGGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAACGT GCCTCTTCCAGTGATTGCTGAGCTGCCTCCCAAAGTGAGCGTCTTCGTCCCACCCCGC GACGGCTTCTTCGGCAACCCCCGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTCA GTCCCCGGCAGATTCAGGTGTCCTGGCTGCGCGAGGGGAAGCAGGTGGGGTCTGGCGT CACCACGGACCAGGTGCAGGCTGAGGCAAAGGAGTCTGGGACCACGACCTACAAGGTG ACCAGCACACTGACCATCAAAGAGAGCGACTGGCTCAGCCAGAGCATGTTCACCTGCC GCGTGGATCACAGGGGCCTGACCTTCCAGCAGAATGCGTCCTCCATGTGTGGCCCCGA TCAAGACACAGCCATCCGGGTCTTCTCCATCCCCCCATCCTTTGCCAGCATCTTCCTC ACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGACAGCGTGA CCATCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTCCGA GAGCCACCCCAATGCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGAC TGGAATTCCGGGGAGAGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCAC TGAAGCAGACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACTT GCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATCACGTGCCTG GTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCAGCCCT TGTCCCCGGAGAAGTATGTGACCAGCGCCCCAATGCCTGAGCCCCAGGCCCCAGGCCG GTACTTCGCCCACAGCATCCTGACCGTGTCCGAAGAGGAATGGAACACGGGGGAGACC TACACCTGCGTGGTGGCCCATGAGGCCCTGCCCAACAGGGTCACCGAGAGGACCGTGG ACAAGTCCACCGGTAAACCCACCCTGTACAACGTGTCCCTGGTCATGTCCGACACAGC TGGCACCTGCTAC  73 human IgM GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS constant VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF region AA VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPT TYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFA SIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASI CEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESAT ITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWN TGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY  74 J Chain DNA ATGAAGAACCATTTGCTTTTCTGGGGAGTCCTGGCGGTTTTTATTAAGGCTGTTCATG TGAAAGCCCAAGAAGATGAAAGGATTGTTCTTGTTGACAACAAATGTAAGTGTGCCCG GATTACTTCCAGGATCATCCGTTCTTCCGAAGATCCTAATGAGGACATTGTGGAGAGA AACATCCGAATTATTGTTCCTCTGAACAACAGGGAGAATATCTCTGATCCCACCTCAC CATTGAGAACCAGATTTGTGTACCATTTGTCTGACCTCTGTAAAAAATGTGATCCTAC AGAAGTGGAGCTGGATAATCAGATAGTTACTGCTACCCAGAGCAATATCTGTGATGAA GACAGTGCTACAGAGACCTGCTACACTTATGACAGAAACAAGTGCTACACAGCTGTGG TCCCACTCGTATATGGTGGTGAGACCAAAATGGTGGAAACAGCCTTAACCCCAGATGC CTGCTATCCTGACTAA  75 J Chain AA MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVER NIIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDED SATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD  76 human CD20 MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIM amino acid NGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGK MIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSE KNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSA EEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSP IENDSSP  77 Ritux-IgM CAGGTTCAGCTGCAGCAGCCCGGAGCCGAGCTGGTCAAACCTGGCGCTAGTGTGAAAA heavy chain TGTCATGCAAGGCATCCGGATACACATTCACTAGCTATAACATGCACTGGGTGAAGCA DNA GACCCCCGGCAGGGGTCTGGAGTGGATCGGAGCTATCTACCCCGGCAACGGAGACACA TCTTATAATCAGAAGTTTAAAGGCAAGGCCACCCTGACAGCTGATAAGTCCAGCTCTA CCGCATACATGCAGCTGAGTTCACTGACAAGCGAGGACTCCGCCGTGTACTATTGCGC CCGGTCCACTTACTATGGCGGAGATTGGTATTTCAATGTGTGGGGAGCAGGCACCACA GTCACCGTCTCGAGCGGCAGTGCTAGCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTG AGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTCGCACAGGACTTCCT TCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAACTCTGACATCAGCAGCACC CGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGCCACCTCACAGGTGCTGC TGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAAGTCCAGCA CCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGCTGAGCTGCCTCCC AAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAAGTCCA AGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCTGCG CGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTGAGGCCAAA GAGTCTGGGCCCACGACCTACAAGGTGACCAGCACACTGACCATCAAAGAGAGCGACT GGCTCAGCCAGAGCATGTTCACCTGCCGCGTGGATCACAGGGGCCTGACCTTCCAGCA GAATGCGTCCTCCATGTGTGTCCCCGATCAAGACACAGCCATCCGGGTCTTCGCCATC CCCCCATCCTTTGCCAGCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCA CAGACCTGACCACCTATGACAGCGTGACCATCTCCTGGACCCGCCAGAATGGCGAAGC TGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAATGCCACTTTCAGCGCCGTG GGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAGAGGTTCACGTGCACCG TGACCCACACAGACCTGCCCTCGCCACTGAAGCAGACCATCTCCCGGCCCAAGGGGGT GGCCCTGCACAGGCCCGATGTCTACTTGCTGCCACCAGCCCGGGAGCAGCTGAACCTG CGGGAGTCGGCCACCATCACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCG TGCAGTGGATGCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGACCAGCGCCCC AATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACCGTGTCC GAAGAGGAATGGAACACGGGGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCCTGC CCAACAGGGTCACCGAGAGGACCGTGGACAAGTCCACCGGTAAACCCACCCTGTACAA CGTGTCCCTGGTCATGTCCGACACAGCTGGCACCTGCTACTGA  78 Ritux-IgM QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDT heavy chain SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTT AA VTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISST RGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPP KVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAK ESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAI PPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAV GEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNL RESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVS EEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY-  79 Ritux-light CAAATTGTGCTGTCTCAGAGTCCAGCTATCCTGAGCGCATCTCCCGGAGAGAAGGTGA chain DNA CCATGACATGCAGAGCCTCCAGCTCTGTCTCCTACATCCACTGGTTCCAGCAGAAGCC CGGCTCCTCCCCAAAACCCTGGATCTACGCCACCTCTAACCTGGCTAGTGGTGTGCCT GTCAGGTTTAGTGGATCAGGGTCCGGCACCAGCTACTCTCTGACAATCAGCCGGGTGG AGGCTGAAGACGCCGCTACATACTATTGCCAGCAGTGGACTTCTAATCCCCCTACCTT CGGCGGAGGGACAAAGCTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATC TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATC GGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCG AAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG TTAG  80 Ritux-light QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVP chain AA VRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-  81 1.5.3 -IgM GAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGAGTCCCTGAAGA heavy chain TCTCCTGCAAGGGCTCCGGCTACTCCTTCACCTCCTACTGGATCGGCTGGGTGAGGCA DNA GATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTACCCCGGCGACTCCGACACC AGGTACTCCCCCTCCTTCCAGGGCCAGGTGACCATCTCCGCCGACAAGTCCATCACCA CCGCCTACCTGCAGTGGTCCTCCCTGAAGGCCTCCGACACCGCCATGTACTACTGCGC CAGGCACCCCTCCTACGGCTCCGGCTCCCCCAACTTCGACTACTGGGGCCAGGGCACC CTGGTGACCGTGTCCTCCGGCAGTGCTAGCGCCCCAACCCTTTTCCCCCTCGTCTCCT GTGAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTCGCACAGGACTT CCTTCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAACTCTGACATCAGCAGC ACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGCCACCTCACAGGTGC TGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAAGTCCA GCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGCTGAGCTGCCT CCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAAGT CCAAGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCT GCGCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTGAGGCC AAAGAGTCTGGGCCCACGACCTACAAGGTGACCAGCACACTGACCATCAAAGAGAGCG ACTGGCTCAGCCAGAGCATGTTCACCTGCCGCGTGGATCACAGGGGCCTGACCTTCCA GCAGAATGCGTCCTCCATGTGTGTCCCCGATCAAGACACAGCCATCCGGGTCTTCGCC ATCCCCCCATCCTTTGCCAGCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGG TCACAGACCTGACCACCTATGACAGCGTGACCATCTCCTGGACCCGCCAGAATGGCGA AGCTGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAATGCCACTTTCAGCGCC GTGGGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAGAGGTTCACGTGCA CCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCAGACCATCTCCCGGCCCAAGGG GGTGGCCCTGCACAGGCCCGATGTCTACTTGCTGCCACCAGCCCGGGAGCAGCTGAAC CTGCGGGAGTCGGCCACCATCACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCT TCGTGCAGTGGATGCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGACCAGCGC CCCAATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACCGTG TCCGAAGAGGAATGGAACACGGGGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCC TGCCCAACAGGGTCACCGAGAGGACCGTGGACAAGTCCACCGGTAAACCCACCCTGTA CAACGTGTCCCTGGTCATGTCCGACACAGCTGGCACCTGCTACTGA  82 1.5.3 -IgM EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDT heavy chain RYSPSFQGQVTISADKSITTAYLQWSSLKASDTAMYYCARHPSYGSGSPNFDYWGQGT AA LVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISS TRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELP PKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEA KESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSA VGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLN LRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTV SEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY-  83 1.5.3 light GACATCGTGATGACCCAGACCCCCCTGTCCTCCCCCGTGACCCTGGGCCAGCCCGCCT chain DNA CCATCTCCTGCAGGTCCTCCCAGTCCCTGGTGTACTCCGACGGCAACACCTACCTGTC CTGGCTGCAGCAGAGGCCCGGCCAGCCCCCCAGGCTGCTGATCTACAAGATCTCCAAC AGGTTCTCCGGCGTGCCCGACAGGTTCTCCGGCTCCGGCGCCGGCACCGACTTCACCC TGAAGATCTCCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCGTGCAGGCCAC CCAGTTCCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCT GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCT CTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGT GGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAG CTTCAACAGGGGAGAGTGTTAG  84 1.5.3 light DIVMTQTPLSSPVTLGQPASISCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKISN chain AA RFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQATQFPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-  85 human IgA1 ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQD constant ASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPP region aa TPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGP P01876 PERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHL LPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTT TFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEV DGTCY  86 human IgA2 ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQD constant ASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSL region aa HRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSS P01877 VLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNEL VTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAE DWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY  87 Human MLLFVLTCLLAVFPAISTKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQ Secretory GARGGCITLISSEGYVSSKYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINS Component RGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPV Precursor LVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKN ADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVVVNTLGKRA PAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNE ESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVK AQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNL KVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDE NSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADA APDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSEEQGGSSR ALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGAND NMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVA AEAQDGPQEA  88 human KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSS secretory KYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLL component NDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIR mature LDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLR GSVTFHCALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSF SVVITGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGS VAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTF TVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPC HFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEG WYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKA IQDPR  89 J15ABD ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCC DNA AGGAAGATGAGCGGATCGTGCTGGTGGACAACAAGTGCAAGTGCGCCCGGATCACCTC CCGGATCATCCGGTCCTCCGAGGATCCCAACGAGGACATCGTGGAACGGAACATCAGA ATCATCGTGCCCCTGAACAACCGCGAGAACATCTCCGACCCCACCAGCCCTCTGCGGA CCAGATTCGTGTACCACCTGTCCGACCTGTGCAAGAAGTGCGACCCTACCGAGGTGGA ACTGGACAACCAGATCGTGACCGCCACCCAGTCCAACATCTGCGACGAGGACTCCGCC ACCGAGACATGCTACACCTACGACCGGAACAAGTGCTACACCGCCGTGGTGCCTCTGG TGTACGGCGGCGAGACAAAGATGGTGGAAACCGCCCTGACCCCCGACGCCTGCTATCC TGATGGAGGCGGAGGATCTGGTGGCGGTGGTTCTGGCGGAGGGGGCTCTCAGCACGAT GAGGCCGTGGACGCCAATTCTCTGGCCGAGGCTAAGGTGCTGGCCAACAGAGAGCTGG ATAAGTACGGCGTGTCCGACTACTACAAGAACCTGATCAACAACGCCAAGACCGTGGA AGGCGTGAAGGCCCTGATCGACGAGATCCTGGCTGCCCTGCCTTGA  90 J15ABD MEWSWVFLFFLSVTTGVHSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIR AA IIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSA TETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGGSGGGGSGGGGSQHD EAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP  91 ABD15J ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCC DNA AGCACGATGAGGCCGTGGACGCCAATTCTCTGGCCGAGGCTAAGGTGCTGGCCAACAG AGAGCTGGATAAGTACGGCGTGTCCGACTACTACAAGAACCTGATCAACAACGCCAAG ACCGTGGAAGGCGTGAAGGCCCTGATCGACGAGATCCTGGCTGCCCTGCCTGGAGGCG GAGGATCTGGTGGCGGTGGTTCTGGCGGAGGGGGCTCTCAGGAAGATGAGCGGATCGT GCTGGTGGACAACAAGTGCAAGTGCGCCCGGATCACCTCCCGGATCATCCGGTCCTCC GAGGATCCCAACGAGGACATCGTGGAACGGAACATCAGAATCATCGTGCCCCTGAACA ACCGCGAGAACATCTCCGACCCCACCAGCCCTCTGCGGACCAGATTCGTGTACCACCT GTCCGACCTGTGCAAGAAGTGCGACCCTACCGAGGTGGAACTGGACAACCAGATCGTG ACCGCCACCCAGTCCAACATCTGCGACGAGGACTCCGCCACCGAGACATGCTACACCT ACGACCGGAACAAGTGCTACACCGCCGTGGTGCCTCTGGTGTACGGCGGCGAGACAAA GATGGTGGAAACCGCCCTGACCCCCGACGCCTGCTATCCTGATTGA  92 ABD15J MEWSWVFLFFLSVTTGVHSQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAK AA TVEGVKALIDEILAALPGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSS EDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIV TATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD  93 HSA15J ATGAAATGGGTCACCTTTATCTCCCTGCTGTTCCTGTTCTCCTCCGCCTACTCTCGGG DNA GCGTGTTCAGAAGAGACGCCCACAAATCGGAGGTAGCGCACCGGTTCAAAGACTTGGG AGAAGAAAACTTTAAGGCCCTTGTACTCATTGCGTTTGCGCAGTATTTGCAGCAGTGC CCATTCGAGGACCATGTCAAACTTGTCAACGAAGTGACAGAGTTTGCGAAAACTTGCG TCGCCGACGAATCCGCGGAGAACTGTGACAAGTCGCTGCATACGTTGTTCGGGGATAA GCTCTGTACCGTAGCGACCTTGAGGGAAACTTACGGGGAAATGGCGGACTGTTGCGCT AAGCAGGAGCCGGAACGGAACGAGTGTTTCCTTCAGCATAAGGATGACAACCCCAACC TCCCTAGATTGGTCAGACCCGAAGTGGATGTGATGTGCACAGCATTCCATGACAATGA GGAAACCTTTCTCAAAAAGTATTTGTACGAGATTGCCCGACGACACCCCTATTTCTAC GCTCCCGAGTTGCTCTTCTTCGCGAAACGGTATAAAGCTGCCTTTACTGAATGCTGTC AAGCAGCGGACAAGGCCGCATGCCTCCTTCCCAAATTGGATGAACTCCGCGATGAAGG GAAGGCGTCATCGGCCAAACAGCGGCTTAAGTGCGCATCGCTTCAGAAATTCGGAGAG AGGGCGTTCAAAGCGTGGGCCGTCGCGAGACTGTCGCAGAGATTCCCTAAGGCGGAAT TTGCAGAGGTATCGAAGCTCGTGACAGACCTCACAAAGGTCCACACCGAATGTTGCCA TGGAGACCTGCTTGAGTGCGCCGATGATAGGGCAGACCTCGCAAAGTACATTTGTGAG AATCAGGACAGCATTAGCTCCAAGCTGAAAGAGTGCTGTGAGAAGCCTTTGCTGGAAA AATCCCACTGTATCGCCGAGGTAGAAAACGATGAAATGCCCGCTGATCTTCCCTCGCT GGCGGCAGACTTCGTCGAGTCGAAGGACGTCTGCAAGAATTACGCAGAGGCAAAAGAT GTGTTTCTTGGAATGTTCCTTTATGAGTATGCGAGAAGGCACCCGGATTATTCCGTGG TACTGCTCTTGCGATTGGCGAAAACGTACGAAACAACGCTTGAGAAGTGTTGTGCGGC TGCCGACCCGCATGAGTGCTACGCCAAGGTATTTGATGAGTTTAAACCTCTTGTCGAG GAACCCCAGAATCTTATCAAGCAGAACTGCGAGCTTTTCAAGCAGTTGGGTGAATACA AATTCCAGAACGCGCTTCTGGTGAGGTATACCAAGAAAGTACCTCAAGTCTCAACACC CACACTCGTCGAGGTGTCACGGAACCTCGGGAAAGTAGGGTCGAAGTGCTGTAAACAC CCAGAGGCCAAGCGCATGCCCTGTGCGGAGGACTACCTCTCGGTAGTGTTGAATCAAC TGTGTGTCCTCCACGAAAAGACGCCGGTGTCAGACCGCGTCACAAAGTGCTGCACGGA GAGCCTGGTCAATAGACGCCCCTGCTTCTCAGCGCTGGAGGTGGATGAGACATACGTC CCGAAAGAGTTTAACGCCGAAACGTTTACTTTTCATGCTGATATCTGTACGTTGTCAG AGAAGGAAAGGCAAATCAAGAAACAAACTGCGCTTGTGGAACTGGTGAAGCACAAACC GAAGGCGACTAAGGAACAGCTGAAGGCGGTGATGGATGACTTTGCCGCGTTCGTAGAG AAATGCTGTAAAGCAGACGATAAGGAGACTTGTTTTGCGGAAGAGGGACCTAAACTTG TTGCTGCAAGTCAAGCTGCCTTAGGCTTAGGAGGCGGAGGATCTGGTGGCGGTGGTTC TGGCGGAGGGGGCTCTCAGGAAGATGAGCGGATCGTGCTGGTGGACAACAAGTGCAAG TGCGCCCGGATCACCTCCCGGATCATCCGGTCCTCCGAGGATCCCAACGAGGACATCG TGGAACGGAACATCAGAATCATCGTGCCCCTGAACAACCGCGAGAACATCTCCGACCC CACCAGCCCTCTGCGGACCAGATTCGTGTACCACCTGTCCGACCTGTGCAAGAAGTGC GACCCTACCGAGGTGGAACTGGACAACCAGATCGTGACCGCCACCCAGTCCAACATCT GCGACGAGGACTCCGCCACCGAGACATGCTACACCTACGACCGGAACAAGTGCTACAC CGCCGTGGTGCCTCTGGTGTACGGCGGCGAGACAAAGATGGTGGAAACCGCCCTGACC CCCGACGCCTGCTATCCTGATTAG  94 HSA15J MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQC AA PFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCA KQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGE RAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICE NQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVE EPQNLIKQNCELFKQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKH PEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV PKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVE KCCKADDKETCFAEEGPKLVAASQAALGLGGGGSGGGGSGGGGSQEDERIVLVDNKCK CARTTSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKC DPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALT PDACYPD  95 J15HSA ATGAAGAACCATCTGCTGTTCTGGGGCGTGCTGGCCGTGTTCATCAAGGCCGTGCACG DNA TGAAGGCCCAGGAAGATGAGCGGATCGTGCTGGTGGACAACAAGTGCAAGTGCGCCCG GATCACCTCCCGGATCATCCGGTCCTCCGAGGATCCCAACGAGGACATCGTGGAACGG AACATCAGAATCATCGTGCCCCTGAACAACCGCGAGAACATCTCCGACCCCACCAGCC CTCTGCGGACCAGATTCGTGTACCACCTGTCCGACCTGTGCAAGAAGTGCGACCCTAC CGAGGTGGAACTGGACAACCAGATCGTGACCGCCACCCAGTCCAACATCTGCGACGAG GACTCCGCCACCGAGACATGCTACACCTACGACCGGAACAAGTGCTACACCGCCGTGG TGCCTCTGGTGTACGGCGGCGAGACAAAGATGGTGGAAACCGCCCTGACCCCCGACGC CTGCTATCCTGATGGAGGCGGAGGATCTGGTGGCGGTGGTTCTGGCGGAGGGGGCTCT GACGCCCACAAATCGGAGGTAGCGCACCGGTTCAAAGACTTGGGAGAAGAAAACTTTA AGGCCCTTGTACTCATTGCGTTTGCGCAGTATTTGCAGCAGTGCCCATTCGAGGACCA TGTCAAACTTGTCAACGAAGTGACAGAGTTTGCGAAAACTTGCGTCGCCGACGAATCC GCGGAGAACTGTGACAAGTCGCTGCATACGTTGTTCGGGGATAAGCTCTGTACCGTAG CGACCTTGAGGGAAACTTACGGGGAAATGGCGGACTGTTGCGCTAAGCAGGAGCCGGA ACGGAACGAGTGTTTCCTTCAGCATAAGGATGACAACCCCAACCTCCCTAGATTGGTC AGACCCGAAGTGGATGTGATGTGCACAGCATTCCATGACAATGAGGAAACCTTTCTCA AAAAGTATTTGTACGAGATTGCCCGACGACACCCCTATTTCTACGCTCCCGAGTTGCT CTTCTTCGCGAAACGGTATAAAGCTGCCTTTACTGAATGCTGTCAAGCAGCGGACAAG GCCGCATGCCTCCTTCCCAAATTGGATGAACTCCGCGATGAAGGGAAGGCGTCATCGG CCAAACAGCGGCTTAAGTGCGCATCGCTTCAGAAATTCGGAGAGAGGGCGTTCAAAGC GTGGGCCGTCGCGAGACTGTCGCAGAGATTCCCTAAGGCGGAATTTGCAGAGGTATCG AAGCTCGTGACAGACCTCACAAAGGTCCACACCGAATGTTGCCATGGAGACCTGCTTG AGTGCGCCGATGATAGGGCAGACCTCGCAAAGTACATTTGTGAGAATCAGGACAGCAT TAGCTCCAAGCTGAAAGAGTGCTGTGAGAAGCCTTTGCTGGAAAAATCCCACTGTATC GCCGAGGTAGAAAACGATGAAATGCCCGCTGATCTTCCCTCGCTGGCGGCAGACTTCG TCGAGTCGAAGGACGTCTGCAAGAATTACGCAGAGGCAAAAGATGTGTTTCTTGGAAT GTTCCTTTATGAGTATGCGAGAAGGCACCCGGATTATTCCGTGGTACTGCTCTTGCGA TTGGCGAAAACGTACGAAACAACGCTTGAGAAGTGTTGTGCGGCTGCCGACCCGCATG AGTGCTACGCCAAGGTATTTGATGAGTTTAAACCTCTTGTCGAGGAACCCCAGAATCT TATCAAGCAGAACTGCGAGCTTTTCAAGCAGTTGGGTGAATACAAATTCCAGAACGCG CTTCTGGTGAGGTATACCAAGAAAGTACCTCAAGTCTCAACACCCACACTCGTCGAGG TGTCACGGAACCTCGGGAAAGTAGGGTCGAAGTGCTGTAAACACCCAGAGGCCAAGCG CATGCCCTGTGCGGAGGACTACCTCTCGGTAGTGTTGAATCAACTGTGTGTCCTCCAC GAAAAGACGCCGGTGTCAGACCGCGTCACAAAGTGCTGCACGGAGAGCCTGGTCAATA GACGCCCCTGCTTCTCAGCGCTGGAGGTGGATGAGACATACGTCCCGAAAGAGTTTAA CGCCGAAACGTTTACTTTTCATGCTGATATCTGTACGTTGTCAGAGAAGGAAAGGCAA ATCAAGAAACAAACTGCGCTTGTGGAACTGGTGAAGCACAAACCGAAGGCGACTAAGG AACAGCTGAAGGCGGTGATGGATGACTTTGCCGCGTTCGTAGAGAAATGCTGTAAAGC AGACGATAAGGAGACTTGTTTTGCGGAAGAGGGACCTAAACTTGTTGCTGCAAGTCAA GCTGCCTTAGGCTTATAG  96 J15HSA MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVER AA NIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDE DSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGGSGGGGSGGGGS DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADES AENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVS KLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCI AEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLR LAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFKQLGEYKFQNA LLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLH EKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQ IKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQ AALGL  97 V15J15ABD ATGGGGTGGTCCTACATTATCCTGTTCCTCGTGGCCACCGCCACTGGCGTGCACTCAC DNA AGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGGT GTCCTGCAAGGCCTCCGGCTACACCTTCATCAGCTACACCATGCACTGGGTGCGACAG GCCCCTGGACAGGGCCTGGAATGGATGGGCTACATCAACCCTAGATCTGGCTACACCC ACTACAACCAGAAGCTGAAGGACAAGGCCACCCTGACCGCCGACAAGTCTGCCTCCAC CGCCTACATGGAACTGTCCTCCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCC AGATCCGCCTACTACGACTACGACGGCTTCGCCTATTGGGGCCAGGGCACCCTCGTGA CAGTGTCTAGCGGTGGCGGAGGATCTGGCGGAGGCGGTAGTGGCGGTGGCGGATCTGA TATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACA ATTACCTGCTCCGCCAGCTCCTCCGTGTCTTACATGAACTGGTATCAGCAGAAGCCCG GCAAGGCCCCCAAGCGGCTGATCTACGACACCTCCAAGCTGGCCTCTGGCGTGCCCTC CAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAG CCCGAGGACTTCGCCACCTACTACTGCCAGCAGTGGTCCTCCAACCCTCCCACCTTTG GCGGAGGCACCAAGGTGGAAATCAAAGGCGGCGGAGGAAGCGGGGGAGGCGGTTCTGG GGGTGGTGGATCTCAGGAAGATGAGCGGATCGTGCTGGTGGACAACAAGTGCAAGTGC GCCCGGATCACCTCCCGGATCATCCGGTCCTCCGAGGATCCCAACGAGGACATCGTGG AACGGAACATCAGAATCATCGTGCCCCTGAACAACCGCGAGAACATCTCCGACCCCAC CAGCCCTCTGCGGACCAGATTCGTGTACCACCTGTCCGACCTGTGCAAGAAGTGCGAC CCTACCGAGGTGGAACTGGACAACCAGATCGTGACCGCCACCCAGTCCAACATCTGCG ACGAGGACTCCGCCACCGAGACATGCTACACCTACGACCGGAACAAGTGCTACACCGC CGTGGTGCCTCTGGTGTACGGCGGCGAGACAAAGATGGTGGAAACCGCCCTGACCCCC GACGCCTGCTATCCTGATGGAGGCGGAGGATCTGGTGGCGGTGGTTCTGGCGGAGGGG GCTCTCAGCACGATGAGGCCGTGGACGCCAATTCTCTGGCCGAGGCTAAGGTGCTGGC CAACAGAGAGCTGGATAAGTACGGCGTGTCCGACTACTACAAGAACCTGATCAACAAC GCCAAGACCGTGGAAGGCGTGAAGGCCCTGATCGACGAGATCCTGGCTGCCCTGCCTT GA  98 V15J15ABD MGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQ AA APGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCA RSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVT ITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQWSSNPPTFGGGTKVEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKC ARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCD PTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTP DACYPDGGGGSGGGGSGGGGSQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINN AKTVEGVKALIDEILAALP  99 V15J15HSA ATGGGGTGGTCCTACATTATCCTGTTCCTCGTGGCCACCGCCACTGGCGTGCACTCAC (K573P) AGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGGT DNA GTCCTGCAAGGCCTCCGGCTACACCTTCATCAGCTACACCATGCACTGGGTGCGACAG GCCCCTGGACAGGGCCTGGAATGGATGGGCTACATCAACCCTAGATCTGGCTACACCC ACTACAACCAGAAGCTGAAGGACAAGGCCACCCTGACCGCCGACAAGTCTGCCTCCAC CGCCTACATGGAACTGTCCTCCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCC AGATCCGCCTACTACGACTACGACGGCTTCGCCTATTGGGGCCAGGGCACCCTCGTGA CAGTGTCTAGCGGTGGCGGAGGATCTGGCGGAGGCGGTAGTGGCGGTGGCGGATCTGA TATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACA ATTACCTGCTCCGCCAGCTCCTCCGTGTCTTACATGAACTGGTATCAGCAGAAGCCCG GCAAGGCCCCCAAGCGGCTGATCTACGACACCTCCAAGCTGGCCTCTGGCGTGCCCTC CAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAG CCCGAGGACTTCGCCACCTACTACTGCCAGCAGTGGTCCTCCAACCCTCCCACCTTTG GCGGAGGCACCAAGGTGGAAATCAAAGGCGGCGGAGGAAGCGGGGGAGGCGGTTCTGG GGGTGGTGGATCTCAGGAAGATGAGCGGATCGTGCTGGTGGACAACAAGTGCAAGTGC GCCCGGATCACCTCCCGGATCATCCGGTCCTCCGAGGATCCCAACGAGGACATCGTGG AACGGAACATCAGAATCATCGTGCCCCTGAACAACCGCGAGAACATCTCCGACCCCAC CAGCCCTCTGCGGACCAGATTCGTGTACCACCTGTCCGACCTGTGCAAGAAGTGCGAC CCTACCGAGGTGGAACTGGACAACCAGATCGTGACCGCCACCCAGTCCAACATCTGCG ACGAGGACTCCGCCACCGAGACATGCTACACCTACGACCGGAACAAGTGCTACACCGC CGTGGTGCCTCTGGTGTACGGCGGCGAGACAAAGATGGTGGAAACCGCCCTGACCCCC GACGCCTGCTATCCTGATGGAGGCGGAGGATCTGGTGGCGGTGGTTCTGGCGGAGGGG GCTCTGACGCCCACAAATCGGAGGTAGCGCACCGGTTCAAAGACTTGGGAGAAGAAAA CTTTAAGGCCCTTGTACTCATTGCGTTTGCGCAGTATTTGCAGCAGTGCCCATTCGAG GACCATGTCAAACTTGTCAACGAAGTGACAGAGTTTGCGAAAACTTGCGTCGCCGACG AATCCGCGGAGAACTGTGACAAGTCGCTGCATACGTTGTTCGGGGATAAGCTCTGTAC CGTAGCGACCTTGAGGGAAACTTACGGGGAAATGGCGGACTGTTGCGCTAAGCAGGAG CCGGAACGGAACGAGTGTTTCCTTCAGCATAAGGATGACAACCCCAACCTCCCTAGAT TGGTCAGACCCGAAGTGGATGTGATGTGCACAGCATTCCATGACAATGAGGAAACCTT TCTCAAAAAGTATTTGTACGAGATTGCCCGACGACACCCCTATTTCTACGCTCCCGAG TTGCTCTTCTTCGCGAAACGGTATAAAGCTGCCTTTACTGAATGCTGTCAAGCAGCGG ACAAGGCCGCATGCCTCCTTCCCAAATTGGATGAACTCCGCGATGAAGGGAAGGCGTC ATCGGCCAAACAGCGGCTTAAGTGCGCATCGCTTCAGAAATTCGGAGAGAGGGCGTTC AAAGCGTGGGCCGTCGCGAGACTGTCGCAGAGATTCCCTAAGGCGGAATTTGCAGAGG TATCGAAGCTCGTGACAGACCTCACAAAGGTCCACACCGAATGTTGCCATGGAGACCT GCTTGAGTGCGCCGATGATAGGGCAGACCTCGCAAAGTACATTTGTGAGAATCAGGAC AGCATTAGCTCCAAGCTGAAAGAGTGCTGTGAGAAGCCTTTGCTGGAAAAATCCCACT GTATCGCCGAGGTAGAAAACGATGAAATGCCCGCTGATCTTCCCTCGCTGGCGGCAGA CTTCGTCGAGTCGAAGGACGTCTGCAAGAATTACGCAGAGGCAAAAGATGTGTTTCTT GGAATGTTCCTTTATGAGTATGCGAGAAGGCACCCGGATTATTCCGTGGTACTGCTCT TGCGATTGGCGAAAACGTACGAAACAACGCTTGAGAAGTGTTGTGCGGCTGCCGACCC GCATGAGTGCTACGCCAAGGTATTTGATGAGTTTAAACCTCTTGTCGAGGAACCCCAG AATCTTATCAAGCAGAACTGCGAGCTTTTCAAGCAGTTGGGTGAATACAAATTCCAGA ACGCGCTTCTGGTGAGGTATACCAAGAAAGTACCTCAAGTCTCAACACCCACACTCGT CGAGGTGTCACGGAACCTCGGGAAAGTAGGGTCGAAGTGCTGTAAACACCCAGAGGCC AAGCGCATGCCCTGTGCGGAGGACTACCTCTCGGTAGTGTTGAATCAACTGTGTGTCC TCCACGAAAAGACGCCGGTGTCAGACCGCGTCACAAAGTGCTGCACGGAGAGCCTGGT CAATAGACGCCCCTGCTTCTCAGCGCTGGAGGTGGATGAGACATACGTCCCGAAAGAG TTTAACGCCGAAACGTTTACTTTTCATGCTGATATCTGTACGTTGTCAGAGAAGGAAA GGCAAATCAAGAAACAAACTGCGCTTGTGGAACTGGTGAAGCACAAACCGAAGGCGAC TAAGGAACAGCTGAAGGCGGTGATGGATGACTTTGCCGCGTTCGTAGAGAAATGCTGT AAAGCAGACGATAAGGAGACTTGTTTTGCGGAAGAGGGACCTAAACTTGTTGCTGCAA GTCAAGCTGCCTTAGGCTTATAG 100 V15J15HSA MGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQ (K573P) APGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCA AA RSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVT ITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQWSSNPPTFGGGTKVEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKC ARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCD PTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTP DACYPDGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFE DHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE PERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPE LLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAF KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQD SISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFL GMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQ NLIKQNCELFKQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCC KADDKETCFAEEGPKLVAASQAALGL 101 V15J15HSA ATGGGGTGGTCCTACATTATCCTGTTCCTCGTGGCCACCGCCACTGGCGTGCACTCAC (wt) DNA AGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGGT GTCCTGCAAGGCCTCCGGCTACACCTTCATCAGCTACACCATGCACTGGGTGCGACAG GCCCCTGGACAGGGCCTGGAATGGATGGGCTACATCAACCCTAGATCTGGCTACACCC ACTACAACCAGAAGCTGAAGGACAAGGCCACCCTGACCGCCGACAAGTCTGCCTCCAC CGCCTACATGGAACTGTCCTCCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCC AGATCCGCCTACTACGACTACGACGGCTTCGCCTATTGGGGCCAGGGCACCCTCGTGA CAGTGTCTAGCGGTGGCGGAGGATCTGGCGGAGGCGGTAGTGGCGGTGGCGGATCTGA TATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACA ATTACCTGCTCCGCCAGCTCCTCCGTGTCTTACATGAACTGGTATCAGCAGAAGCCCG GCAAGGCCCCCAAGCGGCTGATCTACGACACCTCCAAGCTGGCCTCTGGCGTGCCCTC CAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAG CCCGAGGACTTCGCCACCTACTACTGCCAGCAGTGGTCCTCCAACCCTCCCACCTTTG GCGGAGGCACCAAGGTGGAAATCAAAGGCGGCGGAGGAAGCGGGGGAGGCGGTTCTGG GGGTGGTGGATCTCAGGAAGATGAGCGGATCGTGCTGGTGGACAACAAGTGCAAGTGC GCCCGGATCACCTCCCGGATCATCCGGTCCTCCGAGGATCCCAACGAGGACATCGTGG AACGGAACATCAGAATCATCGTGCCCCTGAACAACCGCGAGAACATCTCCGACCCCAC CAGCCCTCTGCGGACCAGATTCGTGTACCACCTGTCCGACCTGTGCAAGAAGTGCGAC CCTACCGAGGTGGAACTGGACAACCAGATCGTGACCGCCACCCAGTCCAACATCTGCG ACGAGGACTCCGCCACCGAGACATGCTACACCTACGACCGGAACAAGTGCTACACCGC CGTGGTGCCTCTGGTGTACGGCGGCGAGACAAAGATGGTGGAAACCGCCCTGACCCCC GACGCCTGCTATCCTGATGGAGGCGGAGGATCTGGTGGCGGTGGTTCTGGCGGAGGGG GCTCTGACGCCCACAAATCGGAGGTAGCGCACCGGTTCAAAGACTTGGGAGAAGAAAA CTTTAAGGCCCTTGTACTCATTGCGTTTGCGCAGTATTTGCAGCAGTGCCCATTCGAG GACCATGTCAAACTTGTCAACGAAGTGACAGAGTTTGCGAAAACTTGCGTCGCCGACG AATCCGCGGAGAACTGTGACAAGTCGCTGCATACGTTGTTCGGGGATAAGCTCTGTAC CGTAGCGACCTTGAGGGAAACTTACGGGGAAATGGCGGACTGTTGCGCTAAGCAGGAG CCGGAACGGAACGAGTGTTTCCTTCAGCATAAGGATGACAACCCCAACCTCCCTAGAT TGGTCAGACCCGAAGTGGATGTGATGTGCACAGCATTCCATGACAATGAGGAAACCTT TCTCAAAAAGTATTTGTACGAGATTGCCCGACGACACCCCTATTTCTACGCTCCCGAG TTGCTCTTCTTCGCGAAACGGTATAAAGCTGCCTTTACTGAATGCTGTCAAGCAGCGG ACAAGGCCGCATGCCTCCTTCCCAAATTGGATGAACTCCGCGATGAAGGGAAGGCGTC ATCGGCCAAACAGCGGCTTAAGTGCGCATCGCTTCAGAAATTCGGAGAGAGGGCGTTC AAAGCGTGGGCCGTCGCGAGACTGTCGCAGAGATTCCCTAAGGCGGAATTTGCAGAGG TATCGAAGCTCGTGACAGACCTCACAAAGGTCCACACCGAATGTTGCCATGGAGACCT GCTTGAGTGCGCCGATGATAGGGCAGACCTCGCAAAGTACATTTGTGAGAATCAGGAC AGCATTAGCTCCAAGCTGAAAGAGTGCTGTGAGAAGCCTTTGCTGGAAAAATCCCACT GTATCGCCGAGGTAGAAAACGATGAAATGCCCGCTGATCTTCCCTCGCTGGCGGCAGA CTTCGTCGAGTCGAAGGACGTCTGCAAGAATTACGCAGAGGCAAAAGATGTGTTTCTT GGAATGTTCCTTTATGAGTATGCGAGAAGGCACCCGGATTATTCCGTGGTACTGCTCT TGCGATTGGCGAAAACGTACGAAACAACGCTTGAGAAGTGTTGTGCGGCTGCCGACCC GCATGAGTGCTACGCCAAGGTATTTGATGAGTTTAAACCTCTTGTCGAGGAACCCCAG AATCTTATCAAGCAGAACTGCGAGCTTTTCAAGCAGTTGGGTGAATACAAATTCCAGA ACGCGCTTCTGGTGAGGTATACCAAGAAAGTACCTCAAGTCTCAACACCCACACTCGT CGAGGTGTCACGGAACCTCGGGAAAGTAGGGTCGAAGTGCTGTAAACACCCAGAGGCC AAGCGCATGCCCTGTGCGGAGGACTACCTCTCGGTAGTGTTGAATCAACTGTGTGTCC TCCACGAAAAGACGCCGGTGTCAGACCGCGTCACAAAGTGCTGCACGGAGAGCCTGGT CAATAGACGCCCCTGCTTCTCAGCGCTGGAGGTGGATGAGACATACGTCCCGAAAGAG TTTAACGCCGAAACGTTTACTTTTCATGCTGATATCTGTACGTTGTCAGAGAAGGAAA GGCAAATCAAGAAACAAACTGCGCTTGTGGAACTGGTGAAGCACAAACCGAAGGCGAC TAAGGAACAGCTGAAGGCGGTGATGGATGACTTTGCCGCGTTCGTAGAGAAATGCTGT AAAGCAGACGATAAGGAGACTTGTTTTGCGGAAGAGGGAAAGAAACTTGTTGCTGCAA GTCAAGCTGCCTTAGGCTTATAG 102 V15J15HSA MGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQ (wt) AA APGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCA RSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVT ITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQWSSNPPTFGGGTKVEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKC ARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCD PTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTP DACYPDGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFE DHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQE PERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPE LLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAF KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQD SISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFL GMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQ NLIKQNCELFKQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCC KADDKETCFAEEGKKLVAASQAALGL 103 S70 IgM HC ATGGACCCCAAGGGCAGCCTGAGCTGGAGAATCCTGCTGTTCCTGAGCCTGGCCTTC DNA GAGCTGAGCTACGGCGAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGC CTGGCGGATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTTACCTTCTCCGACTCCTG GATCCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCTGGATCT CTCCCTACGGCGGCTCTACCTACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTC TGCCGACACCTCCAAGAACACCGCCTACCTGCAGATGAACTCCCTGCGGGCCGAGGA CACCGCCGTGTACTACTGTGCTCGGAGACATTGGCCTGGCGGCTTCGACTATTGGGG CCAGGGCACACTCGTGACCGTGTCTGCTGGAAGTGCTAGCGCCCCAACCCTTTTCCCC CTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTC GCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAACTCTG ACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGCC ACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGT GGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAG TGATTGCTGAGCTGCCTCCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTT CGGCAACCCCCGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCA GATTCAGGTGTCCTGGCTGCGCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACG GACCAGGTGCAGGCTGAGGCCAAAGAGTCTGGGCCCACGACCTACAAGGTGACCAG CACACTGACCATCAAAGAGAGCGACTGGCTCAGCCAGAGCATGTTCACCTGCCGCGT GGATCACAGGGGCCTGACCTTCCAGCAGAATGCGTCCTCCATGTGTGTCCCCGATCA AGACACAGCCATCCGGGTCTTCGCCATCCCCCCATCCTTTGCCAGCATCTTCCTCACCA AGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGACAGCGTGACCA TCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTCCGAGA GCCACCCCAATGCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACT GGAATTCCGGGGAGAGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCAC TGAAGCAGACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACT TGCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATCACGTGC CTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCA GCCCTTGTCCCCGGAGAAGTATGTGACCAGCGCCCCAATGCCTGAGCCCCAGGCCCC AGGCCGGTACTTCGCCCACAGCATCCTGACCGTGTCCGAAGAGGAATGGAACACGG GGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCCTGCCCAACAGGGTCACCGAG AGGACCGTGGACAAGTCCACCGGTAAACCCACCCTGTACAACGTGTCCCTGGTCATG TCCGACACAGCTGGCACCTGCTACTAGTAA 104 S70 IgM HC MDPKGSLSWRILLFLSLAFELSYGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHW AA VRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY CARRHWPGGFDYWGQGTLVTVSAGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLP DSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPN GNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASS MCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNIS ESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLP PAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYF AHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY 105 S70 IgM LC ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC DNA GGAGACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGGGCGACAGA GTGACCATCACCTGTCGGGCCTCTCAGGACGTGTCCACCGCCGTGGCTTGGTATCAG CAGAAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACTCCGCCTCCTTCCTGTACTCCG GCGTGCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACCATCAG CTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACCTGTACCACCCC GCCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCCCCCAG CGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGT GTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACA ACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGA CAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGC ACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAG AGCTTCAACCGGGGCGAGTGCTAA 106 S70 IgM LC METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQ AA KPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKRIVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 107 Y15J QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNN AA KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTV SSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPG QAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGT KVEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIV PLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTY DRNKCYTAVVPLVYGGETKMVETALTPDACYPD 108 V15J MGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQ AA APGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCAR SAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTI TCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARIT SRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQI VTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD

While several embodiments have been provided in the present disclosure, it should be understood that the disclosed systems and methods might be embodied in many other specific forms without departing from the spirit or scope of the present disclosure. The present examples are to be considered as illustrative and not restrictive, and the intention is not to be limited to the details given herein. Various examples of changes, substitutions, and alterations are ascertainable by one skilled in the art and could be made without departing from the spirit and scope disclosed herein.

Claims

1. An antibody comprising: five IgM antibody monomers or five IgG/IgM antibody monomers that form a pentamer; and a modified J-chain, wherein the modified J-chain comprises SEQ ID NO: 1 and a binding moiety that antagonizes a T-cell inhibitory signaling pathway, wherein the binding moiety is attached to SEQ ID NO: 1 at a C- or an N-terminus of the binding moiety, wherein the binding moiety is attached to SEQ ID NO: 1 at a C- or an N-terminus of SEQ ID NO:1 or between cysteine residues 92 and 101 of SEQ ID NO: 1, wherein the IgG/IgM antibody monomers are hybrid antibody monomers which contain an IgM tail-piece at the end of the IgG heavy chain and has the ability to incorporate and form polymers with the modified J-chain, and wherein the binding moiety on the modified J-chain binds to a cell surface protein selected from the group consisting of: CTLA4, PD-1, TIM3, LAG3, BTLA, VISTA and TIGIT.

2. The antibody according to claim 1, wherein the antibody monomers comprise antigen-binding sites that antagonize a T-cell inhibitory signaling pathway.

3. The antibody according to claim 2, wherein the antigen-binding sites bind to a target selected from the group consisting of: PD-1, PD-L1, TIM3 and LAG3.

4. The antibody according to claim 1, wherein the antibody monomers comprise antigen-binding sites that agonize a T-cell stimulatory signaling pathway.

5. The antibody according to claim 4, wherein the antigen-binding sites bind to a target selected from the group consisting of: CD137, OX40, CD40, GITR, CD27 and HVEM.

6. The antibody according to claim 1, wherein the antibody monomers comprise antigen-binding sites that bind to a target selected from the group consisting of: EGFR, HER2, HER3, EpCAM, CEACAM, Gp100, MAGE1 and PD-L1.

7. The antibody according to claim 1, wherein the antibody monomers comprise antigen-binding sites that bind to a target selected from the group consisting of: NY-ESO-1, Sialyl Lewis X antigen and Tn antigen.

8. The antibody according to claim 1, wherein the antibody monomers comprise antigen-binding sites that bind to a cell surface protein on a hematologic cancer cell selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, CD52 and CD70.

9. The antibody according to claim 1, wherein the J-chain binding moiety is attached to SEQ ID NO: 1 by direct or indirect fusion, wherein indirect fusion is via a peptide linker.

10. The antibody according to claim 1, wherein the binding moiety was introduced into the native human J-chain sequence of SEQ ID NO: 1 by chemical or chemo-enzymatic derivatization.

11. The antibody according to claim 1, wherein the binding moiety was introduced into SEQ ID NO: 1 by a cleavable or non-cleavable chemical linker.

12. The antibody according to claim 1, wherein the binding moiety of the modified J-chain is selected from the group consisting of: antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, ligands, and receptors.

13. The antibody according to claim 12, wherein the binding moiety is an antigen-binding fragment of an antibody and is selected from the group consisting of: F(ab′)2, Fab′, Fab, Fv, scFv, and single domain antibody.

14. A pharmaceutical composition for the treatment of cancer, comprising an effective amount of the antibody according to claim 1 and a pharmaceutically acceptable carrier.

Referenced Cited
U.S. Patent Documents
5434340 July 18, 1995 Krimpenfort
5798229 August 25, 1998 Strittmatter
5831034 November 3, 1998 Katinger
5911989 June 15, 1999 Katinger
6165463 December 26, 2000 Platz
6284536 September 4, 2001 Morrison
6476198 November 5, 2002 Kang
6676924 January 13, 2004 Hansen
7074403 July 11, 2006 Goldenberg
7109304 September 19, 2006 Hansen
7138496 November 21, 2006 Hua
7151164 December 19, 2006 Hansen
7238785 July 3, 2007 Govindan
7251164 July 31, 2007 Okhonin
7282567 October 16, 2007 Goldenberg
7300655 November 27, 2007 Hansen
7311912 December 25, 2007 Hein
7312318 December 25, 2007 Hansen
7387773 June 17, 2008 Murray
7402312 July 22, 2008 Rosen
7541440 June 2, 2009 Goldenberg
7601351 October 13, 2009 Rosen
7612180 November 3, 2009 Goldenberg
7709615 May 4, 2010 Irie
7932360 April 26, 2011 Van Berkel
7951378 May 31, 2011 Larrick
8066994 November 29, 2011 Gillies
8114965 February 14, 2012 Maddon
8153125 April 10, 2012 Watkins
8257703 September 4, 2012 Irie
8333971 December 18, 2012 Goldenberg
8337844 December 25, 2012 Carr
8377435 February 19, 2013 Bhat
8871216 October 28, 2014 Chang
9173961 November 3, 2015 Deckert
9409976 August 9, 2016 Teng
9458241 October 4, 2016 Bhat
9938347 April 10, 2018 Wang
9951134 April 24, 2018 Keyt
10351631 July 16, 2019 Keyt
10400038 September 3, 2019 Keyt
10604559 March 31, 2020 Carroll
10618978 April 14, 2020 Keyt
10689449 June 23, 2020 Wang
10899835 January 26, 2021 Baliga
10954302 March 23, 2021 Keyt
10975147 April 13, 2021 Keyt
11401337 August 2, 2022 Baliga
11535664 December 27, 2022 Carroll
11542342 January 3, 2023 Keyt
20020006630 January 17, 2002 Sirbasku
20020168367 November 14, 2002 Larrick
20040005318 January 8, 2004 Davis
20040137001 July 15, 2004 Schreiber
20040156826 August 12, 2004 Dangond
20050003431 January 6, 2005 Wucherpfennig
20050129616 June 16, 2005 Salcedo
20050202026 September 15, 2005 Hiatt
20050287153 December 29, 2005 Dennis
20060063234 March 23, 2006 Jones
20060153854 July 13, 2006 Bhat
20070014720 January 18, 2007 Gazit-Bornstein
20070154469 July 5, 2007 Irie
20070248601 October 25, 2007 Cogne
20070249812 October 25, 2007 Hayasaka
20080044413 February 21, 2008 Hammond
20080145420 June 19, 2008 Simon
20090022738 January 22, 2009 Hofmeister
20090130089 May 21, 2009 Smith
20090291899 November 26, 2009 Ferrante
20100093979 April 15, 2010 Lazar
20100172899 July 8, 2010 Irie
20100184959 July 22, 2010 Guler-Gane
20100279932 November 4, 2010 Ledbetter
20100279939 November 4, 2010 Fries
20110110852 May 12, 2011 Miller
20110129412 June 2, 2011 Gazit-Bornstein
20110318339 December 29, 2011 Smider
20120039870 February 16, 2012 Dolk
20120045432 February 23, 2012 Yu
20120258126 October 11, 2012 Schoeller
20120269830 October 25, 2012 Horowitz
20130095097 April 18, 2013 Blankenship
20130164283 June 27, 2013 Bhat
20130189258 July 25, 2013 Rother
20130280167 October 24, 2013 Rodriguez
20140010809 January 9, 2014 Ledbetter
20140044739 February 13, 2014 Teng
20140088295 March 27, 2014 Smith et al.
20140154252 June 5, 2014 Thompson
20140249044 September 4, 2014 Braz Gonçalves
20150004167 January 1, 2015 Wu
20150038682 February 5, 2015 Tsurushita
20150259420 September 17, 2015 Triebel
20160222132 August 4, 2016 Keyt
20160326233 November 10, 2016 Mondelli
20160368971 December 22, 2016 Keyt
20170183409 June 29, 2017 Keyt
20170283510 October 5, 2017 Keyt
20170320955 November 9, 2017 Wang
20180009897 January 11, 2018 Wang
20180118814 May 3, 2018 Carroll
20180118816 May 3, 2018 Keyt
20180265596 September 20, 2018 Keyt
20190002566 January 3, 2019 Keyt
20190100597 April 4, 2019 Keyt
20190330360 October 31, 2019 Wang
20190330374 October 31, 2019 Wang
20190338031 November 7, 2019 Keyt
20190338040 November 7, 2019 Keyt
20190338041 November 7, 2019 Baliga
20200190190 June 18, 2020 Keyt
20200239572 July 30, 2020 Baliga
20200255546 August 13, 2020 Keyt
20200377577 December 3, 2020 Keyt
20210002353 January 7, 2021 Carroll
20210032357 February 4, 2021 Keyt
20210087273 March 25, 2021 Baliga
20210147567 May 20, 2021 Baliga
20210163600 June 3, 2021 Keyt
20210380701 December 9, 2021 Baliga
20210388098 December 16, 2021 Keyt
20220106398 April 7, 2022 Baliga
20220106399 April 7, 2022 Baliga
20220169751 June 2, 2022 Wang
20220267415 August 25, 2022 Ku
20220289856 September 15, 2022 Amoury
20220306760 September 29, 2022 Keyt
20220340676 October 27, 2022 Baliga
20220372142 November 24, 2022 Baliga
Foreign Patent Documents
697387 March 1996 AU
708301 May 1996 AU
2004297218 June 2005 AU
2358520 July 2000 CA
2662701 October 2008 CA
642585 March 1995 EP
1169352 January 2002 EP
1301541 April 2003 EP
1617870 January 2006 EP
1833848 September 2007 EP
2219458 August 2010 EP
2254592 December 2010 EP
2264163 December 2010 EP
2300498 March 2011 EP
2465871 June 2012 EP
2649184 October 2013 EP
2655415 October 2013 EP
2771361 September 2014 EP
2962103 January 2016 EP
2004525630 August 2004 JP
2005522998 August 2005 JP
1989001975 March 1989 WO
1998030591 July 1998 WO
2001012820 February 2001 WO
2001014424 March 2001 WO
2004110143 December 2004 WO
2004110143 December 2004 WO
2005103081 November 2005 WO
2006052641 May 2006 WO
2006130458 December 2006 WO
2008140477 November 2008 WO
2009013620 January 2009 WO
2009130575 October 2009 WO
2010105256 September 2010 WO
2013061098 May 2013 WO
2013087913 June 2013 WO
2013/120012 August 2013 WO
2013120012 August 2013 WO
2013150138 October 2013 WO
2013188870 December 2013 WO
2014022592 February 2014 WO
2014/124457 August 2014 WO
2014124457 August 2014 WO
2014165093 October 2014 WO
2014/207064 December 2014 WO
2014207064 December 2014 WO
2015037000 March 2015 WO
2015053887 April 2015 WO
2015103072 July 2015 WO
2015120474 August 2015 WO
2015/153912 October 2015 WO
2015151081 October 2015 WO
2015153912 October 2015 WO
2016/118641 July 2016 WO
2016118641 July 2016 WO
2016/141303 September 2016 WO
2016/154593 September 2016 WO
2016141303 September 2016 WO
2016154593 September 2016 WO
2016/168758 October 2016 WO
2016168758 October 2016 WO
2017/059387 April 2017 WO
2017059380 April 2017 WO
2017059387 April 2017 WO
2017196867 November 2017 WO
2018/017761 January 2018 WO
2018/017763 January 2018 WO
2018/017888 January 2018 WO
2018/017889 January 2018 WO
2018017761 January 2018 WO
2018017763 January 2018 WO
2018017888 January 2018 WO
2018017889 January 2018 WO
2018187702 October 2018 WO
2019165340 August 2019 WO
2019169314 September 2019 WO
2020086745 April 2020 WO
2020163646 August 2020 WO
2021030688 February 2021 WO
2021034646 February 2021 WO
2021041250 March 2021 WO
2021055765 March 2021 WO
2021141902 July 2021 WO
2021216756 October 2021 WO
2021231639 November 2021 WO
2022026475 February 2022 WO
2022109023 May 2022 WO
2022178047 August 2022 WO
Other references
  • Cregger (Arch Pathol Lab Med, vol. 130, p. 1026-1030, 2006) (Year: 2006).
  • Rudikoff et al. (Proceedings of the National Academy of Sciences USA, vol. 79, p. 1979-1983, 1982) (Year: 1982).
  • Wang, Y., et al., (2007), “The design, construction and function of a new chimeric anti-CD20 antibody”, Journal of Biotechnology, 129: 726-731.
  • Ammann, J., et al. (2014), “Development and use of IgM/J-Chain Fusion Proteins for Chacterization of Immunoglobulin Superfamily Ligand-Receptor Interactions”, Current Protocols in Protein Science, 19.24.1-19.24.11, Supplement 75.
  • Aggarwal, B., et al., “Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey”, 2012, Blood, vol. 119 (No. 3), pp. 651-665.
  • Ammann, J., et al., (2012), “Detection of weak receptor-ligand interactions using IgM and J-chain-based fusion proteins”, European Journal of Immunology, 42:1354-1356.
  • Andersen, J., et al., “Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding”, 2014, Journal of Biological Chemistry, vol. 289, No. 19, pp. 13492-13502.
  • Azuma, Y., et al., “Recombinant Human Hexamer-Dominant IgM Monoclonal Antibody to Ganglioside GM3 for Treatment of Melanoma”, 2007, Clin Cancer Res, vol. 13 (No. 9), pp. 2745-2750 with correction on article.
  • Bakema et al, “Immunoglobulin A, A next generation of therapeutic antibodies?”, mAbs, Aug. 2011, vol. 3, No. 4, 352-361.
  • Braathen, R., et al. (2002), “The Carboxyl-terminal Domanins of IgA and IgM Direct Isotype-specific Polymerication and Interaction with the Polymeric Immunoglobulin Receptor”, The Journal of Biological Chemistry, vol. 277, No. 45, pp. 42755-42762.
  • Brodska et al., “Correlation of PD-L1 Surface Expression on Leukemia Cells with the Ratio of PD-L1 mRNA Variants and with Electrophoretic Mobility”, Cancer Immunology Research, Aug. 2016, pp. 815-819, vol. 4, No. 10.
  • Cao, Y., et al. , (2011), “Targeting cell surface Beta2-microglobulin by pentameric IgM antibodies”, British Journal of Haematology, 154:111-121.
  • Chintalacharuvu, K., et al., “Hybrid IgA2/IgG1 antibodies with tailor-made effector functions”, 2001, Clin. Immunol., vol. 101 (No. 1), pp. 21-31.
  • Ćirić B., et al. (2009), “Effect of Valency on Binding Properties of the Antihuman IgM Monoclonal Antibody 202”, Hybridoma, vol. 14. No. 6, pp. 537-544.
  • Czajkowsky, D.M., Shao, Z., 2009. The human IgM pentamer is a mushroom-shaped molecule with a flexural bias. PNAS, vol. 106, pp. 14960-14965.
  • Davis, A., (1989), “IgM—Molecular requirements for its assembly and function”, Immunology Today, vol. 10, No. 4, 7 pages.
  • Dennis, M., et al. “Albumin Binding as a General Strategy for Improving the Pharmacokinetics of Proteins”, 2002, J. Biol. Chem. 277, 35035-35043.
  • Dennis, M., et al., (2012), “Transferrin Antibodies into the Brain”, Neuropsychopharmacology Reviews, 37: 302-303.
  • Ducry, L., et al., “Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies”, 2010, Bioconjug. Chem. 21, 5-13.
  • Frutiger, S., et al., 1992, “Disulfide Bond Assignment in Human J Chain and its Covalent Pairing with Immunoglobulin M”, Biochemistry, vol. 31, pp. 12643-12647.
  • Fukushima, N., et al., (2002), “Chacterization of Recombinant Monoclonal IgA Anti-PDC-E2 Autoantibodies Derived From patients with PBC”, Hepatology: 35: 1383-1392.
  • Garcia-Pardo, A., Lamm, M.E., Plaut, A.G., Frangione, B., 1981. J chain is covalently bound to both monomer subunits in human secretory IgA. J. Biol. Chem. 256, 11734-11738.
  • Gibson Josefine, “Anti-PD-L1 for metastatic triple-negative breast cancer.”, The Lancet. Oncology Jun. 2015, (Jun. 2015), vol. 16, No. 6, ISSN 1474-5488, p. e264, XP002765988.
  • Gilmour, J.E.M., et al. (2008), “Effet of the presence or absence of J Chain on expresion of recombinant anti-kell immunoglobulin”, Transfusion Medicine, 18: 167-174.
  • Hopp, J., et al., (2010), “The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein”, vol. 23(11): 827-834.
  • Houghton, J., et al., (2015), “Site-specifically labeled CA19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer”, PNAS, vol. 112, No. 52: 15850-15855.
  • Johansen, F., et al., (2000), “Role of J Cahin in Secretory Immunoglobulin Formation”, Scand. J. Immunol., 52: 240-248.
  • Johansen, F., et al., (2001), “The J Chain Is Essential for Polymeric Ig Receptor-Mediated Epithelial Transport of IgA”, The Journal of Immunology, 167: 5185-5192.
  • Jones, A., et al., (2007), “Blood-Brain Barrier Transport of Therapeutics via Receptor-Mediation”, Pharm Res., 24(9): 1759-1771.
  • Joos, B., et al., 2006, “Long-Term Multiple-Dose Pharmacokinetics of Human Monoclonal Antibodies (MAbs) against Human Immunodeficiency Virus Type 1 Envelope gp120 (MAb 2G12) and gp41 (MAbs 4E10 and 2F5)”, Antimicrob Agents Chemother 50, 1773-1779.
  • Klein, J., et al., (2014,) “Design and characterization of structured protein linkers with differing flexibilities” Protein Eng Des Sel 27, 325-330.
  • Kragten, E., et al., (1995), “Site-specific analysis of the N-Glycans on Murine Polymeric Immunoglobulin A. Using Liquid Chromatography/Electrospray Mass Spectrometry”, vol. 30, 1679-1686.
  • Krugmann, S., et al. (1997), “Structural Requirements for Assembly of Dimeric IgA Probed by Site-Directed Mutagenesis of J Chain and a Cysteine Residue of the α-chain CH2 Domain”, The Journal of Immunology, 159: 244-249.
  • Ksiezak-Reding, H., et al., 1988, “Alz 50, a monoclonal antibody to Alzheimer's disease antigen, cross-reacts with tau proteins from bovine and normal human brain”, J. Biol. Chem. 263, 7943-7947.
  • Lewis, A.K., et al., 2014, “Open and Closed Conformations of the Isolated Transmembrane Domain of Death Receptor 5 Support a New Model of Activation” Biophys J 106, L21-L24.
  • Liedtke, M., et al., 2012. “Phase I trial of a novel human monoclonal antibody mAb216 in patients with relapsed or refractory B-cell acute lymphoblastic leukemia” Haematologica 97, 30-37.
  • Lines, J.L., Pantazi, E., Mak, J., Sempere, L.F., Wang, L., O'Connell, S., Ceeraz, S., Suriawinata, A.A., Yan, S., Ernstoff, M.S., Noelle, R., 2014. VISTA Is an Immune Checkpoint Molecule for Human T Cells. Cancer Res 74, 1924-1932. https://doi.org/10.1158/0008-5472.CAN-13-1504.
  • Liu, X., et al., (2005), “Preliminary study on recombination of J-HNP-1 and its antibacterial effect in vitro” Shijie Huaren Xiaohua Zazhi, 13(22), 2640-2644, English Abstract Only Available.
  • Liu, X., et al., (2005), “Recombinant of J chain-HNP-1 cDNA and the construction of expression vector”,Di-San Junyi Daxue Xuebao, 27(8), 697-699. English Abstract Only Available.
  • Mestecky, J., et al., (1973), “J-chain of polymeric IgA myeloma proteins”, Protides of the Biological Fluids, vol. 20: 279-283 , Abstract Only Available.
  • Meyer et al., “Improved In Vivo Anti-Tumor Effects of IgA-Her2 Antibodies Through Half-Life Extension and Serum Exposure Enhancement by FcRn Targeting”, mAbs, Jan. 2016, pp. 87-98, vol. 8, Issue 1.
  • Mongini, P., et al., (1995), “Human B Cell Activation, Effect of T Cell Cytokines on the Physicochemical Binding Requirements for Achieving Cell cycle Progression Via the Membrane IgM Signaling Pathway”, The Journal of Immunology, 155: 3385-3400.
  • Mordenti, J., et al. (1999), “Intraocular Pharmacokinetics and Safety of a Humanized Monoclonal Antibody in Rabbits after Intravitreal Administration of a Solution or a PLGA Microsphere Formulation”, Toxicological Sciences, 52: 101-106.
  • Mordenti, J., et al., (1999), “Comparisons of the Intraocular Tissue Distribution, Pharmacokinetics, and Safety of 125I-Labeled Full-Length and Fab Antibodies in Rhesus Monkeys Following Intravitreal Administration”, Investigative Pathology, vol. 27, No. 5, pp. 536-544.
  • Mosmann, T.R., et al. (1978), “Modification and Fate of J. Chain in myeloma cells in the presence and absence of polymeric immunoglobulin secretion”, Eur. J Immunol. 8: 84-101.
  • Müller, M. et al., (2012), “Improving the pharmacokinetic properties of biologies by fusion to an anti-HSA shark VNAR domain” MAbs 4, 673-685.
  • Niwa, H., Yamamura, K., Miyazaki, J., 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-199.
  • Nocentini, G., et al., (1997), “A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis”, Proc. Natl. Acad. Sci. U.S.A. 94, 6216-6221.
  • Ofei, F., Hurel, S., Newkirk, J., Sopwith, M., Taylor, R., 1996. Effects of an engineered human anti-TNF-alpha antibody (CDP571) on insulin sensitivity and glycemic control in patients with NIDDM. Diabetes 45, 881-885.
  • Palanichamy Arumugam et al, “Rituximab efficiently depletes increased CD20-expressing T cells in multiple sclerosis patients.”, Journal of Immunology (Baltimore, MD.: 1950) Jul. 15, 2014, (Jul. 15, 2014), vol. 193, No. 2, pp. 580-586.
  • Pardoll, D.M., (2012). “The blockade of immune checkpoints in cancer immunotherapy” Nat. Rev. Cancer 12, 252-264.
  • Paterson, J., et al., (2006), “The differential expression of LCK and BAFF-receptor and their role in apoptosis in human lymphomas”, Haematologica 91: 772-780.
  • Tavolaro, S., (2013), “IgD cross-linking induces gene expression profiling changes and enhances apoptosis in chronic lymphocytic leukemia cells”, Leukemia Research, 37: 455-462.
  • Tussiwand, R., et al., (2012), “BAFF-R expression correlates with positive selection of immature B cells”, European Journal of Immunology, 42: 206-216.
  • Ponders and Richards, “Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes,” J. Mol. Biol. 1987, vol. 193, pp. 775-791.
  • Presta, “Antibody engineering,” Curr. Op. Struct. Biol. 1992, vol. 2, pp. 593-596.
  • Rabbitts et al. “Human immunoglobulin heavy chain genes: evolutionary comparisons of C mu, C delta and C gamma genes and associated switch sequences,” GenBank Accession No. CAB37838, Nucleic Acids Research 1981 vol. 9, No. 18, pp. 4509-4524.
  • Raju et al., “Potential therapeutic roles for antibody mixtures,” Exp. Op. Biol. Ther. 2013, vol. 13, No. 10, pp. 1347-1352.
  • Symersky, J., et al., (2000), “Expression of the recombinant human immunoglobulin J Chain in Escherichia coli”, Molecular Immunology, 37: 133-140.
  • Riechmann et al., “Reshaping human antibodies for therapy,” Nature 1988, vol. 332, pp. 323-329.
  • Roopenian, D., et al. (2007), “FcRn: the neonatal Fc receptor comes of age”, Nature Immunology, vol. 7, 715-725.
  • Seifert, O., et al., (2012), “The IgM CH2 domain as covalently linked homodimerization module for the generation of fusion proteins with dual specificty”, Protein engineering, Design & Selection, vol. 25, No. 10, 603-612.
  • Tabrizi, M., et al. (2010), “Biodistribution Mechanisms of Therapeutic Monoclonal Antibodies in Health and Disease”, The AAPS Journal, vol. 12, No. 1, pp. 33-43.
  • Jeon, H., et al., (2014), “Structure and Cancer Immunotherapy of the B7 Family Member B7x”, Cell Rep., vol. 9(3): 1089-1098.
  • Maekawa, N., et al., (2014), “Expression of PD-L1 on Canine Tumor Cells and Enhancement of IFN-γ Production from Tumor-Infiltrating Cells by PD-L1 Blockade”, PLOS One, vol. 9(6), e98415.
  • Müller, M.R., Saunders, K., Grace, C., Jin, M., Piche-Nicholas, N., Steven, J., O'Dwyer, R., Wu, L., Khetemenee, L., Vugmeyster, Y., Hickling, T.P., Tchistiakova, L., Olland, S., Gill, D., Jensen, A., Barelle, C.J., 2012. Improving the pharmacokinetic properties of biologies by fusion to an anti-HSA shark VNAR domain. MAbs 4, 673-685. https://doi.org/10.4161/mabs.22242.
  • Nocentini, G., Giunchi, L., Ronchetti, S., Krausz, L.T., Bartoli, A., Moraca, R., Migliorati, G., Riccardi, C., 1997. A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis. Proc. Natl. Acad. Sci. U.S.A. 94, 6216-6221.
  • Pardoll, D.M, 2012. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12, 252-264. https://doi.org/10.1038/nrc3239.
  • Paulik, M., Grieco, P., Kim, C., Maxeiner, H.G., Grunert, H.P., Zeichhardt, H., Morré, D.M., Morré, D.J., 1999. Drug-antibody conjugates with anti-HIV activity. Biochem. Pharmacol. 58, 1781-1790.
  • Postow, M.A., Callahan, M.K., Wolchok, J.D., 2015. Immune Checkpoint Blockade in Cancer Therapy. J. Clin. Oncol. 33, 1974-1982. https://doi.org/10.1200/JCO.2014.59.4358.
  • Redwan, E.-R.M., Matar, S.M., Serour, I.A., 2006. Recombinant human J-chain: fix the protein aggregations and yield maximize. Hum Antibodies 15, 95-102.
  • Smith, R.I., Coloma, M.J., Morrison, S.L., 1995. Addition of a mu-tailpiece to IgG results in polymeric antibodies with enhanced effector functions including complement-mediated cytolysis by IgG4. The Journal of Immunology 154, 2226-2236.
  • Sørensen, V., Rasmussen, I.B., Sundvold, V., Michaelsen, T.E., Sandlie, I., 2000. Structural requirements for incorporation of J chain into human IgM and IgA. Int. Immunol. 12, 19-27.
  • Tchoudakova, A., et al., 2009, “High level expression of functional human IgMs in human PER.C6 cells”, MAbs 1, 163-171.
  • Vcelar, B., et al., 2007, “Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data” AIDS 21, 2161-2170.
  • Wood, C.R., et al., 1990, “High level synthesis of immunoglobulins in Chinese hamster ovary cells”. J. Immunol. 145, 3011-3016.
  • Yu, X., et al., 2009, “The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells,” Nat. Immunol. 10, 48-57.
  • Yoo, E., et al., 1999, “Structural Requirements for Polymeric Immunoglobulin Assembly and Association with J Chain” vol. 274, No. 47, pp. 33771-33777.
  • Baliga, R., et al., (2016) “High Avidity Anti-CD20 IgM Antibody for enhanced Complement-Dependent Cell Killing of Low CD20 Expressing Tumor Cells”, Poster Presented at the PEGS Boston Meeting Apr. 25-29, 2016.
  • Cao, Y., et al., (2011), “Targeting cell surface ß2-microglobulin by pentameric IgM antibodies”, Br J Haematol, 154(1): 111-121.
  • Casset, F., et al., (2003), “A peptide mimetic of an anti-CD4 monoclonal antibody by rational design”, Biochemical and biophysical Research Communications, 307: 198-205.
  • Chen, Y., et al., (1999), Selection and Analysis of an Optimized Anti-VEGF Antibody: Crystal Structure of an Affinity-matured Fab in Complex with Antigen, 293: 865-881.
  • De Pascalis, R., et al., (2002), “Grafting of “Abbreviated” Complementarity-Determining Regions Containing specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody”, The Journal of Immunology, 169: 3076-3084.
  • Duramad, O., et al., (2014), “IGM-55.5 a novel monoclonal human recombinant IgM antibody with potent activity against B cell leukemia and lymphoma” IGM Biosciences, Inc.—Research and Development SRI International—Cancer Pharmacology, Stanford—Department of Obstetrics and Gynecology, Abstract No. 645 AACR Annual Meeting, Apr. 5-9, 2014, San Diego CA.
  • Gaetano, N., et al., (2003), “Complement Activation Determines the Therapeutic Activity of Rituximab in Vivo”, The Journal of Immunology, 171: 1581-1587.
  • Johnson, R., et al. (2012) “Biological Activity of Anti-CD20 Multivalent HPMA Copolymer-Fab' Conjugates”, Biomacromolecules, vol. 13, pp. 727-735.
  • Lamminmaki, U., et al., (2001), “Crystal Structure of a Recombinant Anti-estradiol Fab Fragment in Complex with 17ß Estradiol”, The Journal of Biological Chemistry, vol. 276(39): 36687-36694.
  • MacCallum R., et al. (1996), “Antibody-antigen Interactions: Contact Analysis and binding Site Topography”, J. Mol. Bio. 262: 732-745.
  • Padlan, E., et al., (1989), “Structure of an antibody-antigen complex: crystal structure of the HyHel-10 Fab-lysozyme complex;”, Proc. Natl. Acad. Sci. USA, vol. 86: 5938-5942.
  • Pascal, V., et al. (2012) “Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 Target cells”, Haematologica, the Hematology Journal, vol. 97(11), pp. 1686-1694.
  • Randall, T., et al., (1992), “Direct Evidence That J Chain Regulates the Polymeric Structure of IgM in Antibody-secreting B Cells”, The Journal of Biological Chemistry, vol. 267(25), 18002-18007.
  • Rossi, E., et al. (2008) “Novel Designs of Multivalent Anti-CD20 Humanized Antibodies as Improved Lymphoma Therapeutics”, Cancer Res, vol. 68(20), pp. 8384-8392.
  • Rudikoff, S., et al., (1982), “Single amino acid substitution altering antigen-binding specificity”, Proc. Natl. Acad. Sci. USA, 79: 1979-1983.
  • Vajdos, F., et al., (2002), “Comprehensive Functional Maps of the Antigen-binding Site of an Anti-ErbB2 Antibody Obtained with Shotgun Scanning Mutagenesis”, J. Mol. Biol. 320: 415-428.
  • Weiskopf, K., et al. (2013) “Engineered SIRPa Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies”, Science Express, vol. 341, pp. 89-91 with supplemental materials, 36 pages.
  • Woof et al., “Structure and Function Relationships in IgA”, Mucosal Immunology, Nov. 2011, pp. 590-597, vol. 4 No. 6.
  • Wu, H., et al., (1999), “Humanization of a Murine Monoclonal Antibody by simultaneous Optimization of Framework and CDR Residues”, J. Mol. Biol. 294: 151-162.
  • Gibson, “Anti-PD-L1 for metastatic triple-negative breast cancer”, Lancet Oncol., May 2015, pp. e264, vol. 16, No. 6.
  • Harao et al., “Abstract LB-263: Enhancing of CD8+ tumor-infiltrating lymphocyte expansion from triple-negative breast cancer patients using of 41 BB costimulation”, American Association for Cancer Research, Oct. 2014, 2 pages.
  • Ipalanichamy et al., “Rituximab Efficiently Depletes Increased CD20-Expressing T Cells in multiple Sclerosis Patients”, Journal of Immunology, Jul. 15, 2014, pp. 580-586, vol. 193, No. 2.
  • Albrecht, H., et al. (2006), “Recombinant Antibodies: From the Laboratory to the Clinic”, Cancer Biotherapy & Radiopharmaceuticals, vol. 21: 285-304.
  • Mariuzza, R. A., (1987), “The Structural Basis of Antigen-Antibody Recognition”, vol. 16: 139-159.
  • Singer, M., et al., “Genes and Genomes”, Moscow, “Mir”, 1998, vol. 1: 63-64, no translation available, reference is believed to be a standard textbook reference on antibody structure.
  • Ammann, J., et al. (2014), “Development and use of IgM/J-Chain Fusion Proteins for Chacterization of Immunoglobulin Superfamily Ligand-Receptor Interactions”, Current Protocols in Protein Science, Supplement 75.
  • Arnold, J., et al., (2005), “Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin”, The Journal of Biological Chemistry, vol. 280(32): 29080-29087.
  • Bacac, M., et al., (2018), “CD20-TCB with Obinutuzumab Pretreatment as Next-Generation Treatment of Hematologic Malignancies, Clinical Cancer Research, 24(19): 4785-4797, includes supplementary methods,” Binding to CD20- and CD3-expressing cells, 24 additional pages.
  • Baliga, R., et al., (2019), “IGM-2323: High Avidity IgM-based CD20 x CD3 Bispecific Antibody for Enhanced T-Cell Dependent Killing with Minimal Cytokine Release”, Abstract 1574 at American Society of Hematology Annual Meeting, Dec. 7-10, 2019, Orlando, FL.
  • Beers, S., et al., (2010), “Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection”, Blood, 115(25): 5191-5201.
  • Bornstein, G., et al., (2010), “Development of a new fully human anti-CD20 monoclonal antibody for the treatment of B-cell malignancies”, Invest New Drugs, 28:561-574.
  • Bortoletto, N., et al., (2002), “Optimizing anti-CD3 affinity for effective T cell targeting against tumor cells”, Eur. J. Immunol, 32: 3102-3107.
  • Bowles, J., et al., (2006), “Anti-CD20 monoclonal antibody with enhanced affinity for CD16 activates NK cells at lower concentralions and more effectively than rituximab”, Blood, 108(8): 2648-2654.
  • Brüggemann, M., et al., (1987), “Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies”, J. Exp. Med, 166: 1351-1361.
  • Castro, C., et al., (2014), “Putting J chain back on the map: how might its expression define plasma cell development?”, The Journal of Immunology, 193: 3248-3255.
  • Chu, S., et al., (2014), “Immunotherapy with Long-Lived Anti-CD20 x Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-mediated Killing of Human B Cell lines and Circulating and Lymphoid B Cells in Monkeys: A Potential Therapy tor B Cell Lymphomas and Leukemias”, Xencor, Inc., Monrovia, California 91016 USA, Poster.
  • Czuczman, M., et al., (2008), “Acquirement of rituximab resistance in lymphoma cell lines is associated with both global CD20 gene and protein down-regulation regulated at the pretranscriptional and posttranscriptional levels”, Clin Cancer Res, 14(5): 1561-1570.
  • Davis, T., et al., (1999), “Single-agent monoclonal antibody efficacy in bulky non-Hodgkin's lymphoma: results of a phase II trial of rituximab”, J. Clin Oncol. 17: 1851-1857.
  • Davis, T., et al., (2000), “Rituximab anti-CD20 monoclonal antibody therapy in non-Hodgkin's lymphoma: safety and efficacy of re-treatment”, Journal of Clinical Oncology, 18(17): 3135-3143.
  • Ghielmini, M., (2004), “Prolonged treatment with rituximab in patients with follicular lymphoma significantly increases event-free survival and response duration compared with the standard weekly x 4 schedule”, Blood, 103(12): 4416-4423.
  • Hagenbeek, A., et al., (2009), “Evaluation of Ofatumumab, a Novel Human CD20 Monoclonal Antibody, as Single Agent Therapy in Rituximab-Refractory Follicular Lymphoma”, Blood, 114(22): 935, 6 pages.
  • Haidar, JH., et al., (2003), “Loss of CD20 expression in relapsed lymphomas after rituximab therapy”, Eur J. Haematol, 70: 330-332.
  • Harao M., et al. , (2014), “Abstract LB-263: Enhancing of CD8+ tumor-infiltrating lymphocyte expansion from triple-negative breast cancer patients using of 41BB costimulation”, American Associaton for Cancer Research, URL: http://cancerres.aacrjournals.org/content/74/19_Supplement/LB-263, XP002765987.
  • Hensel, F., et al., (2013), “Early development of PAT-SM6 for the treatment of melanoma”, Melanoma Research, 23: 264-275.
  • Hsu, D., et al., (1999), “A humanized anti-CD3 antibody, HuM291, with low mitogenic activity, mediates complete and reversible T-cell depletion in chimpanzees”, Transplantation, 68(4): 545-554.
  • Klimovich, V. B., (2011), “IgM and Its Receptors: Structural and Functional Aspects”, Biochemistry, 76(5): 534-549.
  • Maloney, D., et al., (1997), “IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma”, Blood, 90(6): 2188-2195.
  • Mandikian, D., et al., (2018), “Relative Target Affinities of T Cell-Dependent Bispecific Antibodies Determine Biodistribution in a Solid Tumor Mouse Model”, Mol Cancer Ther., 17(4): 776-785, doi: 10.1158/1535-7163. MCT-17-0657.
  • Marcus, R., et al., (2017), “Obinutuzumab for the First-Line Treatment of Follicular Lymphoma”, N Engl. J. Med. 377(14): 1331-44.
  • Mølhøj, M., et al., (2007), “CD19-/CD3-bispecific antibody of the BiTE class is far superior to tandem diabody with respect to redirected tumor cell lysis”, Molecular Immunology 44: 1935-1943.
  • NCBI Reference Sequence (2018): NP_653247.1,Apr. 17, 2013, Immunoglobulin J chain precursor [Homo sapiens].
  • Miles, M., et al., (1995), “Polymer IgM assembly and secretion in lymphoid and nonlymphoid cell lines: evidence that J chain is required for pentamer IgM synthesis”, Proc. Natl. Acad. Sci. USA, 92:2884-2888.
  • Rasche, L., et al., (2015), “GRP78-directed immunotherapy in relapsed or refractory multiple myeloma—results from a phase 1 trial with the monoclonal immunoglobulin M antibody PAT-SM6”, Haematologica, 100(3): 377-384.
  • Rezvani, A., et al., (2011), “Rituximab Resistance”, Best Pract Res Clin Haematol, 24(2): 203-216.
  • Saber, H., et al., (2017), “An FDA oncology analysis of CD3 bispecific constructs and first-in-human dose selection”, Regulatory Toxicology and Pharmacology, 90: 144-152.
  • Smith, E., et al., (2015), “A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys”, Scientific Reports, 5:17943, 12 pages.
  • Strohl, W., et al., (2015), “Fusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters”, BioDrugs, 29: 215-239.
  • Sun, L., et al., (2015), “Anti-CD20/CD3 T cell-dependent bispecific antibody for the treatment of B cell malignancies”, Immunotherapy, vol. 7(287), 287ra70, 11 pages.
  • Teachey, D., et al., (2013), “Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine directed therapy”, Blood Journal, DOI 10.1182/blood-2013-02-485623.
  • Valley, C., et al., (2014), “Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Induces Death Receptor 5 Networks That Are Highly Organized”, The Journal of Biological chemistry, vol. 287, No. 25, pp. 21265-21278.
  • Van Imhoff, G., et al., (2017), “Ofatumumab Versus Rituximab Salvage Chemoimmunotherapy in Relapsed or Refractory Diffuse Large B-Cell Lymphoma: The ORCHARRD Study”, Journal of Clinical Oncology, 35(5): 544-551.
  • Vitolo, U., et al., (2017), “Obinutuzumab or Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone in Previously Untreated Diffuse Large B-Cell Lymphoma”, Journal of Clinical Oncology, 35(31): 3529-3537.
  • Wing, M., (2008), “Monoclonal Antibody First Dose Cytokine Release Syndromes—Mechanisms and Prediction”, Journal of Immunotoxicology, 5: 11-18.
  • Wu, J., et al., (2015), “Blinatumomab: a bispecific T cell engager (BiTE) antibody against CD19/CD3 for refractory acute lymphoid leukemia”, Journal of Hematology & Oncology, 8:104, DOI 10.1186/s13045-015-0195-4, 7 pages.
  • Request for Supplement Examination as filed on Jun. 18, 2021 with USPTO re U.S. Pat. No. 9,951,134, dated Apr. 24, 2018.
  • Supplemental Examination Certificate dated Aug. 16, 2021 with USPTO re U.S. Pat. No. 9,951,134.
  • U.S. Appl. No. 17/386,397, Specification, Claims, Abstract and Drawings as filed Jul. 27, 2021 with U.S. Patent Office.
  • U.S. Appl. No. 17/635,078, Specification, Claims, Abstract and Drawings as filed Feb. 14, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 17/758,207, Specification, Claims, Abstract and Drawings as filed Jun. 29, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 17/761,428, Specification, Claims, Abstract and Drawings as filed Mar. 17, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 17/806,339, Specification, Claims, Abstract and Drawings as filed Jun. 10, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 17/812,614, Specification, Claims, Abstract and Drawings as filed Jul. 14, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 17/996,760, Specification, Claims, Abstract and Drawings as filed Oct. 20, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 18/052,388, Specification, Claims, Abstract and Drawings as filed Nov. 3, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 17/998,307, Specification, Claims, Abstract and Drawings as filed Nov. 9, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 18/054,776, Specification, Claims, Abstract and Drawings as filed Nov. 11, 2022 with U.S. Patent Office.
  • U.S. Appl. No. 18/055,340, Specification, Claims, Abstract and Drawings as filed Nov. 14, 2022 with U.S. Patent Office.
Patent History
Patent number: 11639389
Type: Grant
Filed: Sep 30, 2016
Date of Patent: May 2, 2023
Patent Publication Number: 20190185570
Assignee: IGM Biosciences, Inc. (Mountain View, CA)
Inventors: Bruce A. Keyt (Hillsborough, CA), Leonard G. Presta (San Francisco, CA), Ramesh Baliga (Redwood City, CA)
Primary Examiner: Laura B Goddard
Assistant Examiner: Sarah A Alsomairy
Application Number: 15/764,870
Classifications
Current U.S. Class: Immunoglobulin, Antiserum, Antibody, Or Antibody Fragment, Except Conjugate Or Complex Of The Same With Nonimmunoglobulin Material (424/130.1)
International Classification: C07K 16/28 (20060101); C07K 16/30 (20060101); C07K 16/46 (20060101); C07K 16/18 (20060101);